Systems and methods for determining a state of fluidization and/or a state of mixing

ABSTRACT

Apparatus and methods are disclosed for treating a sample by selectively controlling sonic energy and/or selectively controlling the location of the sample relative to the sonic energy.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.10/777,014, filed Feb. 11, 2004, which is a continuation of U.S. patentapplication Ser. No. 09/830,473, filed on Apr. 27, 2001, now U.S. Pat.No. 6,719,449 which is the U.S. national phase of International (PCT)Patent Application Ser. No. PCT/US99/25274, filed on Oct. 28, 1999, andpublished in English on May 4, 2000, which claims the benefit of andpriority to U.S. provisional application Ser. Nos. 60/105,933, filedOct. 28, 1998; 60/110,460, filed Dec. 1, 1998; 60/119,500, filed Feb.10, 1999; 60/143,440, filed Jul. 13, 1999; and 60/148,279, filed Aug.11, 1999, the entirety of each of the disclosures of these provisionalapplications being incorporated by reference herein.

TECHNICAL FIELD

The present invention generally relates to the field of controlled sonicenergy emitting devices for treating material, particularly biologicalmaterial.

BACKGROUND OF THE INVENTION

Ultrasonics have been utilized for many years for a variety ofdiagnostic, therapeutic, and research purposes. The acoustic physics ofultrasonics is well understood; however, the biophysical, chemical, andmechanical effects are generally only empirically understood. Some usesof sonic or acoustic energy in materials processing include“sonication,” an unrefined process of mechanical disruption involvingthe direct immersion of an unfocused ultrasound source emitting energyin the kilohertz (“kHz”) range into a fluid suspension of the materialbeing treated. Accordingly, the sonic energy often does not reach atarget in an effective dose because the energy is scattered, absorbed,and/or not properly aligned with the target. There are also specificclinical examples of the utilization of therapeutic ultrasound (e.g.,lithotripsy) and of diagnostic ultrasound (e.g., fetal imaging).However, ultrasonics have heretofore not been controlled to provide anautomated, broad range, precise materials processing or reaction controlmechanism.

SUMMARY OF THE INVENTION

The present invention relates to apparatus and methods for selectivelyexposing a sample to sonic energy, such that the sample is exposed toproduce a desired result such as, but without limitation, heating thesample, cooling the sample, fluidizing the sample, mixing the sample,stirring the sample, disrupting the sample, permeabilizing a componentof the sample, enhancing a reaction in the sample, and sterilizing thesample. For example, altering the permeability or accessibility of amaterial, especially labile biological materials, in a controlled mannercan allow for manipulation of the material while preserving theviability and/or biological activity of the material. In anotherexample, mixing materials or modulating transport of a component into orout of materials, in a reproducible, uniform, automated manner, can bebeneficial. According to one embodiment of the system, sample processingcontrol includes a feedback loop for regulating at least one of sonicenergy location, pulse pattern, pulse intensity, and absorbed dose ofthe ultrasound. The system can be automated. In one embodiment, theultrasonic energy is in the megahertz (MHz) frequency range, in contrastto classical sonic processing which typically employs ultrasonic energyin the kilohertz (kHz) frequency range.

When ultrasonic energy interacts with a complex biological or chemicalsystem, the acoustic field often becomes distorted, reflected, anddefocused. The net effect is that energy distribution becomesnon-uniform and/or defocused compared to the input. Non-uniform reactionconditions can limit reaction applications to non-critical processes,such as bulk fluid treatment where temperature gradients within a sampleare inconsequential. However, some of the non-uniform aspects are highlydeleterious to samples, such as extreme temperature gradients thatdamage sample integrity. For example, in some instances, the hightemperature would irreversibly denature target proteins. As aconsequence, many potential applications of ultrasound, especiallybiological applications, are limited to specific, highly-specializedapplications, such as lithotripsy and diagnostic imaging, because of thepotentially undesirable and uncontrollable aspects of ultrasound incomplex systems.

Typically, when ultrasound is applied to a bulk biological samplesolution, such as for the extraction of intracellular constituents fromtissue, the treatment causes a complex, heterogeneous, mixture ofsub-events that vary during the course of a treatment dose. In otherwords, the ultrasonic energy may be partitioned between various states.For example, the energy may directly treat a sample or the energy mayspatially displace a target moiety and shift the target out of theoptimal energy zone. Additionally or alternatively, the energy mayresult in interference that reflects the acoustic energy. For example, a“bubble shield” occurs when a wave front of sonic energy createscavitation bubbles that persist until the next wave front arrives, suchthat the energy of the second wave front is at least partially blockedand/or reflected by the bubbles. Still further, larger particles in thesample may move to low energy nodes, thereby leaving the smallerparticles in the sample with more dwell-time in the high energy nodes.In addition, the sample viscosity, temperature, and uniformity may varyduring the ultrasonic process, resulting in gradients of theseparameters during processing. Accordingly, current processes aregenerally random and non-uniform, especially when applied to in vitroapplications, such as membrane permeabilization, hindering the use ofultrasound in high throughput applications where treatmentstandardization from one sample to the next is required.

Processing samples containing labile material, in particular biologicalmaterial, is still largely a manual process, and poorly adapted tohigh-throughput sample processing required for applications such aspharmaceutical and agricultural genomics. For example, except forisolated or exposed cells, the insertion of a nucleic acid into asample, for temporary or permanent transformation, is stillsubstantially manual. Most transformation techniques have been developedfor a small subset of materials, which typically have only a singleplasma membrane separating their interior from the environment. Thesemembranes may be permeabilized using detergents, salts, osmotic shock,or simple freeze-thawing. Thus, materials such as viruses, culturedcells, and bacteria and protists, such as yeast, which have been treatedto prevent the formation of cell walls, can be transfected by any of anumber of standard methods. For example, transfection can be undertakenwith vectors including viruses that bind to plasma membranes for directtransport, and can be undertaken in a direct transfection with “naked”DNA that is often coated with cationic lipids or polymers or that is inthe presence of chemical or biochemical membrane permeabilizing agents.

Moreover, many biological materials of interest have supportingstructures, and are significantly harder to permeabilize or otherwise toaccess the plasma membrane with macromolecular agents or viruses. Thesupporting structures range from simple cell walls, as in yeast, tocomplex protein and glycoprotein structures, as in animal tissue, totenacious and only slowly degradable polysaccharide structures, as inplants and insects, to physically durable mineralized supports, as indiatoms and bone. In all of these “hard” materials, physical disruptionof the supporting matrices is required typically to precede or accompanytransfection or other nucleic acid insertion to allow reliableintroduction of extracellular components.

Sonication has been used to break up difficult materials such as planttissue. Sonication, typically implemented by vibration of a probe atfrequencies of 10,000 Hz or higher, creates shearing forces within aliquid sample. However, the resultant shear is not readily controlled,so that when sufficient energy is applied to disrupt a supportingmatrix, the shear will also tend to destroy fragile intracellularstructures. Indeed, sonication is routinely used to randomly shear DNAin solution into small fragments. Such fragmentation limits theusefulness of these techniques for many purposes, and particularly fortransfection, which requires a viable cell to be successful.

The present invention addresses these problems and provides apparatusand methods for the non-contact treatment of samples with ultrasonicenergy, using a focused beam of energy. The frequency of the beam can bevariable and can be in the range of about 100 kHz to 100 MHz, morepreferably 500 kHz to 10 MHz. For example, the present invention cantreat samples with ultrasonic energy while controlling the temperatureof the sample, by use of computer-generated complex wavetrains, whichmay further be controlled by the use of feedback from a sensor. Theacoustic output signal, or wavetrain, can vary in any or all offrequency, intensity, duty cycle, burst pattern, and pulse shape. Inanother example, the present invention can treat samples with ultrasonicenergy when the samples are in an array, and individual samples in thearray may be treated differentially or identically. Moreover, thistreatment can be undertaken automatically under computer control. Inanother example, the present invention can treat samples with ultrasonicenergy in a uniform way over the entire sample, by the relative movementof the sample and the focus of the beam, in any or all of two or threedimensions.

The apparatus and methods of the present invention can be controlled bya computer program. In one embodiment, the sequence of actions taken bythe computer is predetermined. Such embodiments can be useful inhigh-speed, high-volume processing. In another embodiment, the processesare enhanced with a program that uses feedback control to modify ordetermine the actions thereof, using techniques including algorithmicprocessing of input, the use of lookup tables, and similar integrationdevices and processes.

A feedback control mechanism, in connection with any of accuracy,reproducibility, speed of processing, control of temperature, provisionof uniformity of exposure to sonic pulses, sensing of degree ofcompletion of processing, monitoring of cavitation, and control of beamproperties (including intensity, frequency, degree of focusing,wavetrain pattern, and position), can enhance certain embodiments of thepresent invention. A variety of sensors or sensed properties may beappropriate for providing input for feedback control. These propertiescan include sensing of temperature of the sample; sonic beam intensity;pressure; bath properties including temperature, salinity, and polarity;sample position; and optical or visual properties of the samples. Theseoptical properties may include apparent color, emission, absorption,fluorescence, phosphorescence, scattering, particle size, laser/Dopplerfluid and particle velocities, and effective viscosity. Sample integrityor comminution can be sensed with a pattern analysis of an opticalsignal. Any sensed property or combination thereof can serve as inputinto a control system. The feedback can be used to control any output ofthe system, for example beam properties, sample position, and treatmentduration.

The samples can be treated in any convenient vessel or container.Vessels can be sealed for the duration of the treatment to preventcontamination of the sample or of the environment. Arrays of vessels canbe used for processing large numbers of samples. These arrays can bearranged in one or more high throughput configurations. Examples includemicrotiter plates, typically with a temporary sealing layer to close thewells, blister packs, similar to those used to package pharmaceuticalssuch as pills and capsules, and arrays of polymeric bubbles, similar tobubble wrap, preferably with a similar spacing to typical microtiterwells. The latter are described in more detail below.

The treatment, which may be performed or enhanced by use of ultrasonicwavetrains, include any unit operation which is susceptible to beingimplemented or is enhanced by sonic waves or pulses. In particular,these results include lysing, extracting, permeabilizing, stirring ormixing, comminuting, heating, fluidizing, sterilizing, catalyzing, andselectively degrading. Sonic waves may also enhance filtration, fluidflow in conduits, and fluidization of suspensions. Processes of theinvention may be synthetic, analytic, or simply facilitative of otherprocesses such as stirring.

Any sample is potentially suitable for processing by the techniques andapparatuses of the invention. For example, any material that includesbiological organisms or material derived therefrom is suitable. Manychemicals can be processed more efficiently, particularly in small-scaleor combinatorial reactions or assays, with the processes of theinvention, including remote, non-contact mixing or stirring. Physicalobjects, such as mineral samples and particulates including sands andclays, also can be treated with the present invention.

According to the present invention, several aspects of the invention canenhance the reproducibility and/or effectiveness of particulartreatments using ultrasonic energy in in vitro applications, wherereproducibility, uniformity, and precise control are desired. Theseaspects include the use of feedback, precise focusing of the ultrasonicenergy, monitoring and regulating of the acoustic waveform (includingfrequency, amplitude, duty cycle, and cycles per burst), positioning ofthe reaction vessel relative to the ultrasonic energy so that the sampleis uniformly treated, controlling movement of the sample relative to thefocus of ultrasonic energy during a processing step, and/or controllingthe temperature of the sample being treated, either by the ultrasonicenergy parameters or through the use of temperature control devices suchas a water bath. A treatment protocol can be optimized, using one or acombination of the above variables, to maximize, for example, shearing,extraction, permeabilization, comminution, stirring, or other processsteps, while minimizing undesirable thermal effects.

In one embodiment of the invention, high intensity ultrasonic energy isfocused on a reaction vessel, and “real time” feedback relating to oneor more process variables is used to control the process. In anotherembodiment, the process is automated and is used in a high throughputsystem, such as a 96-well plate, or a continuous flowing stream ofmaterial to be treated, optionally segmented.

Minimization of unwanted interference with the pattern of appliedultrasonic energy is another feature of the invention. For example,ultrasonic energy applied to a sample in a reaction vessel has thepotential to directly interact with the target sample, or to reflectfrom bubbles or other effects from a previous cycle of ultrasoundapplication and not interact with the target, or to miss the targetbecause of spatial separation or mismatch. Minimization of interferenceis especially beneficial for remote, automated, sterile processing ofsmall amounts of target material, for example, 10 mg of a biopsy tissue.By minimizing the reflections and optimizing spatial positioning, theultrasonic energy is more efficiently utilized and controlled. Theprocess can be standardized to obtain reproducibility by presettingconditions such as waveform and positioning, by a feedback signal andfeedback-based control to maintain preset performance target parameters,or by a combination of these methods.

In certain embodiments, the processing system can include a highintensity transducer that produces acoustic energy when driven by anelectrical or optical energy input; a device or system for controllingexcitation of the transducer, such as an arbitrary waveform generator,an RF amplifier, and a matching network for controlling parameters suchas time, intensity, and duty cycle of the ultrasonic energy; apositioning system such as a 2-dimensional (x, y) or a 3-dimensional (x,y, z) positioning system that can be computer controlled to allowautomation and the implementation of feedback from monitoring; atemperature sensor; a device for controlling temperature; one or morereaction vessels; and a sensor for detecting, for example, optical,radiative, and/or acoustic signatures.

Vessels containing the samples can be sealed during the processing, andhence can be sterile throughout, or after, the procedure. Moreover, theuse of focused ultrasound allows the samples in the vessels to beprocessed, including processing by stirring, without contacting thesamples, even when the vessels are not sealed.

The processes have a variety of applications, including, but withoutlimitation, extraction, permeabilization, mixing, comminuting,sterilization, flow control, and reacting. For example, mixing in avessel can be achieved with temperature fluctuations controlled towithin about plus or minus one degree Celsius. More precise control ispossible, if required. In another example, labile biological materialscan be extracted from plant materials without loss of activity or theuse of harsh solvents. In other applications, complex cells can bepermeabilized and molecules such as nucleotide molecules can beintroduced into the cells using the process of the invention. Otherapplications include modulating binding reactions that are useful inseparations, biological assays, and hybridization reactions.

One aspect of the invention includes an apparatus for processing asample using sonic energy. The apparatus includes a sonic energy sourcefor emitting sonic energy; a holder for the sample, the sample movablerelative to the emitted sonic energy; and a processor for controllingthe sonic energy source and location of the sample according to apredetermined methodology, such that the sample is selectively exposedto sonic energy to produce a desired result. The desired result can beheating the sample, cooling the sample, fluidizing the sample, mixingthe sample, stirring the sample, disrupting the sample, increasingpermeability of a component of the sample, enhancing a reaction withinthe sample, and/or sterilizing the sample. Also, the desired result canbe an in vitro or an ex vivo treatment.

This aspect and other aspects of the invention can include any or all ofthe following features. The apparatus can further include a feedbacksystem connected to the processor for monitoring at least one conditionto which the sample is subjected during processing, such that theprocessor controls at least one of the sonic energy source and thelocation of the sample in response to the at least one condition. Thefeedback system can include a sensor for monitoring the at least onecondition. The apparatus can further include a temperature control unitfor controlling temperature of the sample, and the processor can controlthe temperature control unit. The apparatus can further include apressure control unit for controlling pressure to which the sample isexposed, and the processor controls the pressure control unit. The sonicenergy source can include a transducer. The transducer can focus thesonic energy and can include at least one piezoelectric element, anarray of piezoelectric elements, an electrohydraulic element, amagnetostrictive element, an electromagnetic transducer, a chemicalexplosive element, and/or a laser-activated element. A piezoelectricelement can include a spherical transmitting surface oriented such thatthe focal axis is oriented vertically or in any other predetermineddirection. The holder can support a sample container for containing thesample. The sample container can be a membrane pouch, thermopolymerwell, polymeric pouch, hydrophobic membrane, microtiter plate,microtiter well, test tube, centrifuge tube, microfuge tube, ampoule,capsule, bottle, beaker, flask, and/or capillary tube. The samplecontainer can form multiple compartments and can include a rupturablemembrane for transferring a fraction of the sample away from the holder.The apparatus can further include a device for moving the sample from afirst location to a second location; such as a stepper motor. Theapparatus can also include an acoustically transparent material disposedbetween the sonic energy source and the holder. The sample can flowthrough a conduit. The sonic energy source can generate sonic energy attwo or more different frequencies, optionally in the form of a serialwavetrain. The wavetrain can include a first wave component and adifferent second wave component. Alternatively or additionally, thewavetrain can include about 1000 cycles per burst at about a 10% dutycycle at about a 500 mV amplitude.

Another aspect of the invention relates to a method for processing asample with sonic energy. The method includes the steps of exposing thesample to sonic energy and controlling at least one of the sonic energyand location of the sample relative to the sonic energy according to apredetermined methodology, such that the sample is selectively exposedto sonic energy to produce a desired result. The desired result can beheating the sample, cooling the sample, fluidizing the sample, mixingthe sample, stirring the sample, disrupting the sample, increasingpermeability of a component of the sample, enhancing a reaction withinthe sample, and/or sterilizing the sample. Also, the desired result canbe an in vitro or an ex vivo treatment. This aspect or any of the otheraspects of the invention can include any or all of the followingfeatures. The method can further include the steps of sensing at leastone condition to which the sample is subjected during processing andaltering at least one of the sonic energy and the location of the samplein response to the sensed condition. During the sensing step, the sensedcondition can be temperature, pressure, an optical property, an alteredchemical, an acoustic signal, and/or a mechanical occurrence. During thealtering step, the characteristic of the sonic energy that is alteredcan be waveform, duration of application, intensity, and/or duty cycle.The method can further include the step of controlling temperature ofthe sample and can further include the step of controlling pressure towhich the sample is exposed. During the step of exposing the sample tosonic energy, the sonic energy can be generated by spark dischargesacross a gap, laser pulses, piezoelectric pulses, electromagnetic shockwaves, electrohydraulic shock waves, electrical discharges into aliquid, and/or chemical explosives. The sonic energy can be focused onthe sample. The sample can contain a cell, and the method can furthercomprise the step of introducing a material into the cell. The materialcan be a polymer, an amino acid monomer, an amino acid chain, a protein,an enzyme, a nucleic acid monomer, a nucleic acid chain, a saccharide, apolysaccharide, an organic molecule, an inorganic molecule, a vector, aplasmid, and/or a virus. The method can further include the step ofextracting a component of the sample. During the controlling step, atleast one characteristic of the sonic energy is controlled, thatcharacteristic being waveform, duration of application, intensity, orduty cycle. The method can further include the step of the sampleflowing through a conduit. The sonic energy can include at least twodifferent frequencies, optionally in the form of a wavetrain. Thewavetrain can include a first wave component and a different second wavecomponent Alternatively or additionally, the wavetrain can include about1000 cycles per burst at about a 10% duty cycle at about a 500 mVamplitude.

BRIEF DESCRIPTION OF THE DRAWINGS

The invention, in accordance with preferred and exemplary embodiments,together with further advantages thereof, is more particularly describedin the following detailed description, taken in conjunction with theaccompanying drawings.

In the drawings, like reference characters generally refer to the sameparts throughout the different views. Also, the drawings are notnecessarily to scale, emphasis instead generally being placed uponillustrating principles of the invention.

FIG. 1 is a schematic illustration of one embodiment of the apparatusaccording to the invention;

FIG. 2 is a schematic illustration of one example of sonic energycontrol showing sine waves at a variable amplitude and frequency;

FIG. 3 is a schematic illustration of one example of an intra-samplepositioning (dithering) profile showing height, height step, and radius;

FIG. 4A is a schematic illustration of a vertical-sided treatmentvessel;

FIG. 4B is a schematic illustration of a conical treatment vessel;

FIG. 4C is a schematic illustration of a curved treatment vessel;

FIGS. 5A-5C are schematic illustrations of several embodiments of atreatment vessel with a combination of an upper and lower member andsamples in the vessels prior to treatment;

FIG. 6A is a schematic illustration of a treatment vessel positionedover a collection container prior to transferring the contents of thevessel to the container;

FIG. 6B is a schematic illustration of a treatment vessel positionedover a collection container after transferring some of the contents ofthe vessel to the container.

FIG. 7 is a schematic illustration of an in-line fluid treatment methodin accordance with an alternative embodiment of the invention;

FIG. 8 is a graph depicting change in sample temperature as a functionof duty cycle at 500 mV and 750 mV, in one embodiment of the invention;

FIG. 9 is a schematic illustration of an embodiment of the inventionwith a microtiter plate containing samples, such that one of the wellsof the microtiter plate is positioned at the focus point of sonicenergy;

FIG. 10 describes certain features and specifications related toperformance, consumables, procedure for treatment, and mechanicalcomponents of a system according to certain embodiments of theinvention;

FIG. 11 describes certain features and specifications related toinstrument control, user interface, electrical, and associated equipmentof a system according to certain embodiments of the invention;

FIG. 12 describes certain characteristics and functionality of operatingsoftware related to general functions, display functions, sonic energycontrol, and target/source positioning of a system according to certainembodiments of the invention; and

FIG. 13 describes certain additional characteristics and functionalityof operating software related to target/source positioning andtemperature control of a system according to certain embodiments of theinvention.

DETAILED DESCRIPTION OF THE INVENTION

“Sonic energy” as used herein is intended to encompass such terms asacoustic energy, acoustic waves, acoustic pulses, ultrasonic energy,ultrasonic waves, ultrasound, shock waves, sound energy, sound waves,sonic pulses, pulses, waves, or any other grammatical form of theseterms, as well as any other type of energy that has similarcharacteristics to sonic energy. “Focal zone” or “focal point” as usedherein means an area where sonic energy converges and/or impinges on atarget, although that area of convergence is not necessarily a singlefocused point. As used herein, the terms “microplate,” “microtiterplate,” “microwell plate,” and other grammatical forms of these termscan mean a plate that includes one or more wells into which samples maybe deposited. As used herein, “nonlinear acoustics” can mean lack ofproportionality between input and output. For example, in ourapplication, as the amplitude applied to the transducer increases, thesinusoidal output loses proportionality such that eventually the peakpositive pressure increases at a higher rate than the peak negativepressure. Also, water becomes nonlinear at high intensities, and in aconverging acoustic field, the waves become more disturbed as theintensity increases toward the focal point. Nonlinear acousticproperties of tissue can be useful in diagnostic and therapeuticapplications. As used herein, “acoustic streaming” can mean generationof fluid flow by acoustic waves. The effect can be non-linear. Bulkfluid flow of a liquid in the direction of the sound field can becreated as a result of momentum absorbed from the acoustic field. Asused herein, “acoustic microstreaming” can mean time-independentcirculation that occurs only in a small region of the fluid around asource or obstacle for example, an acoustically driven bubble in a soundfield. As used herein, “acoustic absorption” can refer to acharacteristic of a material relating to the material's ability toconvert acoustic energy into thermal energy. As used herein, “acousticimpedance” can mean a ratio of sound pressure on a surface to sound fluxthrough the surface, the ratio having a reactance and a resistancecomponent. As used herein, “acoustic lens” can mean a system or devicefor spreading or converging sounds waves. As used herein, “acousticscattering” can mean irregular and multidirectional reflection anddiffraction of sound waves produced by multiple reflecting surfaces, thedimensions of which are small compared to the wavelength, or by certaindiscontinuities in the medium through which the wave is propagated.

I. Apparatus and Methods for Ultrasonic Treatment

In certain embodiments, the apparatus includes a source of sonic energy,a sensor for monitoring the energy or its effect, and a feedbackmechanism coupled with the source of sonic energy to regulate the energy(for example, voltage, frequency, pattern) for transmitting ultrasonicenergy to a target. Devices for transmission may include detection andfeedback circuits to control one or more of losses of energy atboundaries and in transit via reflection, dispersion, diffraction,absorption, dephasing and detuning. For example, these devices cancontrol energy according to known loss patterns, such as beam splitting.Sensors can detect the effects of ultrasonic energy on targets, forexample, by measuring electromagnetic emissions, typically in thevisible, IR, and UV ranges, optionally as a function of wavelength.These effects include energy dispersion, scattering, absorption, and/orfluorescence emission. Other measurable variables include electrostaticproperties such as conductivity, impedance, inductance, and/or themagnetic equivalents of these properties. Measurable parameters alsoinclude observation of physical uniformity pattern analysis, andtemporal progression uniformity across an assembly of treatment vessels,such as a microtiter plate.

As shown in FIG. 1, one or more sensors coupled to a feedback controlresults in more focused, specific, or controlled treatment than thatpossible using current methods typical in the art. The feedbackmethodology can include fixed electronic elements, a processor, acomputer, and/or a program on a computer. The electronic elements,processor, computer, and/or computer program can in turn control any ofa variety of adjustable properties to selectively expose a sample tosonic energy in a given treatment. These properties can includemodulation of the ultrasonic beam in response to a detected effect.Modifiable ultrasonic wave variables can include intensity, duty cycle,pulse pattern, and spatial location. Typical input parameters that cantrigger an output can include change in level of signal, attainment ofcritical level, plateauing of effect, and/or rate of change. Typicaloutput actions can include sonic input to sample, such as frequency,intensity, duty cycle; stopping sample movement or sonic energy; and/ormoving beam within a sample or to the next sample.

More particularly, FIG. 1 depicts an electronically controlledultrasonic processing apparatus 100 that includes an ultrasoundtreatment system and associated electronics 200, a positioning system300 for the sample target 800 being treated, and a control system 400which controls, generates, and modulates the ultrasound signal andcontrols the positioning system 300 in a predetermined manner that mayor may not include a feedback mechanism. The source of sonic energy 230and the target 800 being treated for example, a sample, multiplesamples, or other device are arranged in a fluid bath 600, such aswater, such that the source of sonic energy 230 is oriented towards thetarget 800. The target 800 may be positioned proximate the surface ofthe fluid bath 600, above the source of sonic energy 230, all beingcontained within a sample processing vessel 500. Any of a multitude ofsensors 700 for measuring processing parameters can be arranged in orproximate to the fluid bath 600. A temperature control unit 610 may beused to control the temperature of the fluid in the fluid bath 610. Anoverpressure system 900 can control, for example, cavitation, bymaintaining a positive pressure on the target 800 and may be adjusted,in a predetermined manner that may or may not include feedbackprocessing, by a target pressure controller 910 that is connected to thecontrol system 400.

An ultrasound acoustic field 240 can be generated by the sonic energysource 230, for example, a focused piezoelectric ultrasound transducer,into the fluid bath 600. According to one embodiment, the sonic energysource 230 can be a 70 mm diameter spherically focused transducer havinga focal length of 63 mm, which generates an ellipsoidal focal zoneapproximately 2 mm in diameter and 6 mm in axial length when operated ata frequency of about 1 MHz. The sonic energy source 230 is positioned sothat the focal zone is proximate the surface of the fluid bath 600. Thesonic energy source 230 can be driven by an alternating voltageelectrical signal generated electronically by the control system 400.

The positioning system 300 can include at least one motorized linearstage 330 that allows the target to be positioned according to aCartesian coordinate system. The positioning system 300 may position andmove the target 800 relative to the source 230 in three dimensions (x,y, z) and may optionally move either or both of the target 800 and thesonic energy source 230. The positioning system 300 can move target 800during and as part of the treatment process and between processes, aswhen multiple samples or devices within the target 800 are to beprocessed in an automated or high-throughput format. The positioningsystem 300 may position or move the target 800 in a plane transverse tothe focal axis of the sonic energy source 230 (x and y axes). Thepositioning system 300 can position and move the target 800 along thefocal axis of the sonic energy source 230 and lift or lower the target800 from or into the fluid bath 600 (z axis). The positioning system 300can also position the sonic energy source 230 and any or all of thesensors 700 in the fluid bath 600 along the focal axis of the sonicenergy source 230, if the sensors 700 are not affixed in the water bath600, as well as lift, lower, or otherwise move the sonic energy source230. The positioning system 300 also can be used to move other devicesand equipment such as detection devices and heat exchange devices fromor into the fluid bath 600 (z axis). The linear stages of thepositioning mechanism 330 can be actuated by stepper motors (not shown),which are driven and controlled by electrical signals generated by thecontrol system 400, or other apparatus known to those skilled in theart.

The control system 400 can include a computer 410 and a userinput/output device or devices 420 such as a keyboard, display, printer,etc. The control system is linked with the ultrasound treatment system200 to drive the sonic energy source 230, with the positioning system300 to drive the stepper motors described above, with one or moresensors 700 to detect and measure process conditions and parameters, andwith one or more controllers, such as the target pressure controller910, to alter conditions to which the target 800 is exposed. A fluidbath controller 610 could also be linked with the control system 400 toregulate temperature of the fluid bath 600. The user interface 420allows an operator to design and specify a process to be performed upona sample. In this regard, the ultrasound treatment system 200 caninclude an arbitrary waveform generator 210 that drives an RF amplifier220, such that the sonic energy source 230 receives an input The outputsignal of the RF amplifier 220 may be conditioned by an impedancematching network and input to the sonic energy source 230. The computer410 also drives and controls the positioning system 300 through, forexample, a commercially available motion control board 310 and steppermotor power amplifier device 320.

The control system 400 can generate a variety of useful alternatingvoltage waveforms to drive the sonic energy source 230. For instance, ahigh power “treatment” interval consisting of about 5 to 1,000 sinewaves, for example, at 1.1 MHz, may be followed by a low power“convection mixing” interval consisting of about 1,000 to 1,000,000 sinewaves, for example, at the same frequency. “Dead times” or quiescentintervals of about 100 microseconds to 100 milliseconds, for example,may be programmed to occur between the treatment and convection mixingintervals. A combined waveform consisting of concatenated treatmentintervals, convection mixing intervals, and dead time intervals may bedefined by the operator or selected from a stored set of preprogrammedwaveforms. The selected waveform may be repeated a specified number oftimes to achieve the desired treatment result. Measurable or discernibleprocess attributes such as sample temperature, water bath temperature,intensity of acoustic cavitation, or visible evidence of mixing in thesample processing vessel 500, may be monitored by the control system 400and employed in feedback loop to modify automatically the treatmentwaveform during the treatment process. This modification of thetreatment waveform may be a proportional change to one or more of thewaveform parameters or a substitution of one preprogrammed waveform foranother. For instance, if the sample temperature deviates excessivelyduring treatment from a set-point temperature due to absorbed acousticenergy, the control system 400 may proportionally shorten the treatmentinterval and lengthen the convection mixing interval in response to theerror between the actual and target sample temperatures. Or,alternatively, the control system 400 may substitute one predeterminedwaveform for another. The control system 400 may be programmed toterminate a process when one or more of the sensors 700 signal that thedesired process result has been attained.

The control system 400 controls and drives the positioning system 300with the motion control board 310, power amplifier device 320, andmotorized stage 330, such that the target 800 can be positioned or movedduring treatment relative to the source 230 to selectively expose thetarget 800 to sonic energy, described more fully below.

Various aspects of the embodiment of FIG. 1 and of components of theembodiment shown in FIG. 1, as well as other embodiments with the same,similar, and/or different components, are more fully described below.

A. Transducer

In certain embodiments, the sonic energy source 230, for example, anultrasound transducer or other transducer, produces acoustic waves inthe “ultrasonic” frequency range. Ultrasonic waves start at frequenciesabove those that are audible, typically about 20,000 Hz or 20 kHz, andcontinue into the region of megahertz (MHz) waves. The speed of sound inwater is about 1000 meters per second, and hence the wavelength of a1000 Hz wave in water is about a meter, typically too long for specificfocusing on individual areas less than one centimeter in diameter,although usable in non-focused field situations. At 20 kHz thewavelength is about 5 cm, which is effective in relatively smalltreatment vessels. Depending on the sample and vessel volume, preferredfrequencies may be higher, for example, about 100 kHz, about 1 MHz, orabout 10 MHz, with wavelengths, respectively, of approximately 1.0, 0.1,and 0.01 cm. In contrast, for conventional sonication, including sonicwelding, frequencies are typically approximately in the tens of kHz, andfor imaging, frequencies are more typically about 1 MHz and up to about20 MHz. In lithotripsy, repetition rates of pulses are fairly slow,being measured in the hertz range, but the sharpness of the pulsesgenerated give an effective pulse wavelength, or in this case, pulserise time, with frequency content up to about 100 to about 300 MHz, or0.1-0.3 gigahertz (GHz).

The frequency used in certain embodiments of the invention also will beinfluenced by the energy absorption characteristics of the sample or ofthe treatment vessel, for a particular frequency. To the extent that aparticular frequency is better absorbed or preferentially absorbed bythe sample, it may be preferred. The energy can be delivered in the formof short pulses or as a continuous field for a defined length of time.The pulses can be bundled or regularly spaced.

A generally vertically oriented focused ultrasound beam may be generatedin several ways. For example, a single-element piezoelectric transducer,such as those supplied by Sonic Concepts, Woodinville, Wash., that canbe a 1.1 MHz focused single-element transducer, can have a sphericaltransmitting surface that is oriented such that the focal axis isvertical. Another embodiment uses a flat unfocused transducer and anacoustic lens to focus the beam. Still another embodiment uses amulti-element transducer such as an annular array in conjunction withfocusing electronics to create the focused beam. The annular arraypotentially can reduce acoustic sidelobes near the focal point by meansof electronic apodizing, that is by reducing the acoustic energyintensity, either electronically or mechanically, at the periphery ofthe transducer. This result can be achieved mechanically by partiallyblocking the sound around the edges of a transducer or by reducing thepower to the outside elements of a multi-element transducer. Thisreduces sidelobes near the energy focus, and can be useful to reduceheating of the vessel. Alternatively, an array of small transducers canbe synchronized to create a converging beam. Still another embodimentcombines an unfocused transducer with a focusing acoustic mirror tocreate the focused beam. This embodiment can be advantageous at lowerfrequencies when the wavelengths are large relative to the size of thetransducer. The axis of the transducer of this embodiment can behorizontal and a shaped acoustic mirror used to reflect the acousticenergy vertically and focus the energy into a converging beam.

In certain embodiments, the focal zone can be small relative to thedimensions of the treatment vessel to avoid heating of the treatmentvessel. In one embodiment, the focal zone has a radius of approximately1 mm and the treatment vessel has a radius of at least about 5 mm.Heating of the treatment vessel can be reduced by minimizing acousticsidelobes near the focal zone. Sidelobes are regions of high acousticintensity around the focal point formed by constructive interference ofconsecutive wavefronts. The sidelobes can be reduced by apodizing thetransducer either electronically, by operating the outer elements of amulti-element transducer at a lower power, or mechanically, by partiallyblocking the acoustic waves around the periphery of a single elementtransducer. Sidelobes may also be reduced by using short bursts, forexample in the range of about 3 to about 5 cycles in the treatmentprotocol.

The transducer can be formed of a piezoelectric material, such as apiezoelectric ceramic. The ceramic may be fabricated as a “dome”, whichtends to focus the energy. One application of such material's is insound reproduction; however, as used herein, the frequency is generallymuch higher and the piezoelectric material would be typicallyoverdriven, that is driven by a voltage beyond the linear region ofmechanical response to voltage change, to sharpen the pulses. Typically,these domes have a longer focal length than that found in lithotripticsystems, for example, about 20 cm versus about 10 cm focal length.Ceramic domes can be damped to prevent ringing. The response is linearif not overdriven. The high-energy focus of one of these domes istypically cigar-shaped. At 1 MHz, the focal zone is about 6 cm long andabout 2 cm in diameter for a 20 cm dome, or about 15 mm long and about 3mm wide for a 10 cm dome. The peak positive pressure obtained from suchsystems is about 1 MPa (mega Pascal) to about 10 MPa pressure, or about150 PSI (pounds per square inch) to about 1500 PSI, depending on thedriving voltage.

The wavelength, or characteristic rise time multiplied by sound velocityfor a shock wave, is in the same general size range as a cell, forexample about 10 to about 40 micron. This effective wavelength can bevaried by selection of the pulse time and amplitude, by the degree offocusing maintained through the interfaces between the source and thematerial to be treated, and the like.

In certain embodiments, the focused ultrasound beam is orientedvertically in a water tank so that the sample may be placed at or nearthe free surface. The ultrasound beam creates shock waves at the focalpoint. In an embodiment to treat industry standard microplates whichhold a plurality of samples in an array, a focal zone, defined as havingan acoustic intensity within about 6 dB of the peak acoustic intensity,is formed around the geometric focal point. This focal zone has adiameter of approximately 2 mm and an axial length of about 6 mm.

Ceramic domes are adaptable for in vitro applications because of theirsmall size. Also, systems utilizing ceramic domes can be produced atreasonable cost. They also facilitate scanning the sonic beam focus overa volume of liquid, by using microactuators which move a retainingplatform to which the sample treatment vessel is attached.

Another source of focused pressure waves is an electromagnetictransducer and a parabolic concentrator, as is used in lithotripsy. Theexcitation tends to be more energetic, with similar or larger focalregions. Strong focal peak negative pressures of about −16 MPa have beenobserved. Peak negative pressures of this magnitude provide a source ofcavitation bubbles in water, which can be desirable in an extractionprocess.

The examples described below use a commercial ultrasonic driver using apiezoelectric ceramic, which is stimulated by application of fluctuatingvoltages across its thickness to vibrate and so to produce acousticwaves. These may be of any of a range of frequencies, depending on thesize and composition of the driver. Such drivers are used inlithotripsy, for example, as well as in acoustic speakers and inultrasound diagnostic equipment, although without the control systems asdescribed herein.

These commercially-available drivers have a single focus. Therefore, totreat, for example, to stir, an entire microplate with such a device, itis typically necessary to sequentially position or step each well at thefocus of the driver. Because stirring time is brief, the stepping of a96 well plate can be accomplished in approximately two minutes or lesswith simple automatic controls, as described below. It is contemplatedthat this time can be shortened.

It also is possible to make multi-focal drivers by making piezoelectricdevices with more complex shapes. Modulators of the acoustic fieldattached to an existing piezoelectric driver can also produce multiplefoci. These devices can be important for obtaining rapid throughput ofmicroplates in a high density format, such as the 1534-well format.

B. Drive Electronics and Waveform Control.

One treatment protocol can include variable acoustic waveforms combinedwith sample motion and positioning to achieve a desired effect. Theacoustic waveform of the transducer has many effects, including:acoustic microstreaming in and near cells due to cavitation, that isflow induced by, for example, collapse of cavitation bubbles; shockwaves due to nonlinear characteristics of the fluid bath; shock wavesdue to cavitation bubbles; thermal effects, which lead to heating of thesample, heating of the sample vessel, and/or convective heat transferdue to acoustic streaming; flow effects, causing deflection of samplematerial from the focal zone due to shear and acoustic pressure, as wellas mixing due to acoustic streaming, that is flow induced by acousticpressure; and chemical effects.

The treatment protocol can be optimized to maximize energy transferwhile minimizing thermal effects. The treatment protocol also caneffectively mix the contents of the treatment vessel, in the case of aparticulate sample suspended in a liquid. Energy transfer into thesample can be controlled by adjusting the parameters of the acousticwave such as frequency, amplitude, and cycles per burst. Temperaturerise in the sample can be controlled by limiting the duty cycle of thetreatment and by optimizing heat transfer between the treatment vesseland the water bath. Heat transfer can be enhanced by making thetreatment vessel with thin walls, of a relatively highly thermallyconductive material, and/or by promoting forced convection by acousticstreaming in the treatment vessel and in the fluid bath in the proximityof the treatment vessel. Monitoring and control of temperature isdiscussed in more detail below.

For example, for a cellular disruption and extraction treatment, anexample of an effective energy waveform is a high amplitude sine wave ofabout 1000 cycles followed by a dead time of about 9000 cycles, which isabout a 10% duty cycle, at a frequency of about 1.1 MHz. The sine waveelectrical input to the transducer typically results in a sine waveacoustic output from the transducer. As the focused sine waves convergeat the focal point, they can become a series of shock waves due to thenonlinear acoustic properties of the water or other fluid in the bath.This protocol treats the material in the focal zone effectively duringthe “on” time. As the material is treated, it typically is expelled fromthe focal zone by acoustic shear and streaming. New material circulatesinto the focal zone during the “off” time. This protocol can beeffective, for example, for extracting the cellular contents of groundor particulate leaf tissue, while causing minimal temperature rise inthe treatment vessel.

Further advantage in disruption and other processes may be gained bycreating a high power “treat” interval 10 alternating with a low power“mix” interval 14, as shown schematically in FIG. 2. More particularly,in this example, the “treat” interval 10 utilizes a sine wave that has atreatment frequency 18, a treatment cycles-per-burst count 26, and atreatment peak-to-peak amplitude 22. The “mix” interval 14 has a mixfrequency 20, a mix cycles-per-burst count 28 and a lower mixpeak-to-peak amplitude 24. Following each of the intervals 10, 14 is adead time 12, 16. Of course, these relationships are merely one exampleof many, where one interval in considered to be high power and oneinterval is considered to be low power, and these variables and otherscan be altered to produce more or less energetic situations.Additionally, the treat function or interval and the mix function orinterval could emit from different or multiple transducers in the sameapparatus, optionally emitting at different frequencies.

High power/low power interval treatments can allow multiple operationsto be performed, such as altering permeability of components, such ascells, within the sample followed by subsequent mixing of the sample.The treat interval can maximize cavitation and bioeffects, while the mixinterval can maximize mixing within the treatment vessel and/or generateminimal heat. Adding a longer, high power “super-mix” intervaloccasionally to stir up particles that are trapped around the peripheryof the treatment vessel can provide further benefits. This “super-mix”interval generates additional heat, so it is programmed to treatinfrequently during the process, for example, every few seconds.Additionally, dead times between the mix and treat intervals, duringwhich time substantially no energy is emitted from the sonic energysource, can allow fresh material to circulate into the energy focal zoneof the target.

As discussed below, moving the sample vessel during treatment relativeto the source, so that the focal zone moves within the treatment vessel,can further enhance the process. For example, target motion through thefocal zone can resuspend material in the sample that may have clumped orbecome trapped around the periphery of the treatment vessel. A similarimprovement can be achieved by traversing or “dithering” the treatmentvessel relative to the focal zone, described more fully below withrespect to FIG. 3. Dithering can become increasingly advantageous as thesample treatment vessel becomes significantly larger than the focalzone.

The waveform of focused sound waves can be a single shock wave pulse, aseries of individual shock wave pulses, a series of shock wave bursts ofseveral cycles each, or a continuous waveform. Incident waveforms can befocused directly by either a single element, such as a focused ceramicpiezoelectric ultrasonic transducer, or by an array of elements withtheir paths converging to a focus. Alternatively, multiple foci can beproduced to provide ultrasonic treatment to multiple treatment zones,vessels, or wells.

Reflected waveforms can be focused with a parabolic reflector, such asis used in an “electromagnetic” or spark-gap type shock-wave generator.Incident and reflected waveforms can be directed and focused with anellipsoidal reflector such as is used in an electrohydraulic generator.Waveforms also can be channeled.

The waveform of the sound wave typically is selected for the particularmaterial being treated. For example, to enhance cavitation, it can bedesirable to increase the peak negative pressure following the peakpositive pressure. For other applications, it can be desirable to reducecavitation but maintain the peak positive pressure. This result can beachieved by performing the process in a pressurized chamber at a slightpressure above ambient. For example, if the waveform generated has apeak negative pressure of about −5 MPa, then the entire chamber may bepressurized to about 10 MPa to eliminate cavitation from occurringduring the process. Liquid to be treated can be pressurized on a batchor a continuous basis.

A variety of methods of generating waves can be used. In lithotripsy,for example, “sharp” shock waves of high intensity and short durationare generated. Shock waves may be generated by any method that isapplicable to a small scale. Such methods include spark dischargesacross a known gap; laser pulses impinging on an absorptive orreflective surface; piezoelectric pulses; electromagnetic shock waves;electrohydraulic shock waves created by electrical discharges in aliquid medium; and chemical explosives. In the case of explosives,microexplosives in wells in a semiconductor-type chip can be fabricatedin which the wells are individually addressable. Also, amagnetostrictive material can be exposed to a magnetic field, and it canexpand and/or contract such that the material expansion/contractioncreates sonic energy.

Continuous sinusoidal sound waves can be generated by any process thatis appropriate for focusing on a small scale. For example, ceramicpiezoelectric elements may be constructed into dome shapes to focus thesound wave into a point source. In addition, two or more shock waves maybe combined from the same source, such as piezoelectric elementsarranged in mosaic form, or from different sources, such as anelectromagnetic source used in combination with a piezoelectric source,to provide a focused shock wave.

Typically, the shock wave is characterized by a rapid shock front with apositive peak pressure in the range of about 15 MPa, and a negative peakpressure in the range of about negative 5 MPa. This waveform is of abouta few microseconds duration, such as about 5 microseconds. If thenegative peak is greater than about 1 MPa, cavitation bubbles may form.Cavitation bubble formation also is dependent upon the surroundingmedium. For example, glycerol is a cavitation inhibitive medium, whereasliquid water is a cavitation promotive medium. The collapse ofcavitation bubbles forms “microjets” and turbulence that impinge on thesurrounding material.

The waves are applied to the samples either directly, as for example,piezoelectric pulses, or via an intervening medium. This medium can bewater or other fluid. An intervening medium also can be a solid, such asa material which is intrinsically solid or a frozen solution. Waves alsocan be applied through a container, such as a bottle, bag, box, jar, orvial.

For maximum control, and particularly for well-by-well mixing, a focusedacoustic pulse is useful. When a pulse is emitted from a curved sourcewith an elliptical profile, then the emitted acoustic waves or pulsesfocus in a small region of maximum intensity. The location of the focuscan be calculated or determined readily by experiment. The diameter ofthe focal zone can be of the same general size as or smaller than thediameter of the treatment vessel. Then, mixing energy can be provided toeach well for a repeatable amount of time, providing uniform mixing ofeach sample.

C. X-Y-Z Cartesian Positioning System.

In certain embodiments, the sample is not only moved into positionrelative to the transducer initially, but positioned during treatment toinsure uniform treatment of the sample, where the sample is kept wellsuspended during treatment. As used herein, x and y axes define a planethat is substantially horizontal relative to ground and/or a base of anapparatus of the invention, while the z axis lies in a plane that issubstantially vertical relative to the ground and/or the base of anapparatus and perpendicular to the x-y plane.

One positioning scheme is termed “dithering,” which entails slightlyvarying the position of the sample relative to the source which canoccur by moving the sample through the focal zone in several ways. Forexample, but without limitation, the sample can be moved in a circle, oroval, or other arcuate path with a certain radius 30 and moved a certaindistance 34 in certain increments or steps 32, as depicted schematicallyin FIG. 3. These movements can vary between treatment cycles or during aparticular treatment cycle and have several effects. First, ditheringthe sample position sweeps the focal zone through the volume of thesample treatment vessel or device, treating material that is notinitially in the focal zone. In addition, varying the location of theacoustic focus within the vessel tends to make treatment, and theresulting heating, more uniform within each sample.

Certain embodiments include drive electronics and devices forpositioning of the sample(s). In one embodiment, the positioningsequence, optionally including dithering, and the treatment pulse trainare pre-programmed, for example in a computer, and are executedautomatically. The driver electronics and positioners can be linkedthrough the control system to sensors so that there is “real time”feedback of sensor data to the control system during treatment in orderto adjust the device(s) for positioning the sample and prevent localizedheating or cavitation. The drive electronics can include a waveformgenerator matching network, an RF switch or relay, and a radio frequency(RF) amplifier, for safety shutdown.

The positioning system can include a three axis Cartesian positioningand motion control system to position the sample treatment vessel or anarray of sample treatment vessels relative to the ultrasound transducer.The “x” and “y” axes of the Cartesian positioning system allow eachsample in an array of samples, such as an industry standard microplate,to be brought into the focal zone for treatment. Alternativeconfigurations may employ a combination of linear and rotary motioncontrol elements to achieve the same capabilities as the three axisCartesian system. Alternative positioning systems may be constructed ofself-contained motor-driven linear or rotary motion elements mounted toeach other and to a base plate to achieve two- or three-dimensionalmotion.

As used in the examples, stepper motors, such as those available fromEastern Air Devices, located in Dover, N.H., drive linear motionelements through lead screws to position the sample. The stepper motorsare driven and controlled by means of LabVIEW software controlling aValueMotion stepper motor control board available from NationalInstruments located in Austin, Tex. The output signals from the controlboard are amplified by a nuDrive multi-axis power amplifier interface,also available from National Instruments, to drive the stepper motors.

The computer controlled positioning system can be programmed tosequentially move any defined array of multiple samples into alignmentwith the focal zone of the ultrasound transducer. If temperature riseduring treatment is an issue, the samples in a multi-sample array can bepartially treated and allowed to cool as the positioning systemprocesses the other samples. This can be repeated until all the sampleshave been treated fully.

The positioning system also can move the sample treatment vesselrelative to the focal point during treatment to enhance the treatment orto treat a sample that is large relative to the focal zone. By sweepingthe sample slowly in a circular or other motion during treatment, clumpsof material around the periphery of the treatment vessel may be brokenup advantageously. In addition, x-y dithering may prevent a “bubbleshield” from forming and blocking cavitation in the sample treatmentvessel. The x-y dithering may also enhance treatment of samplesuspensions that have a high viscosity or become more viscous duringtreatment and do not mix well. The sample position may also be ditheredvertically in the Z axis. This may be advantageous in a deep treatmentvessel where the depth is substantially larger than the axial dimensionof the focal zone, in order to treat the entire contents of thetreatment vessel or to resuspend larger sample fragments which have sunkto the bottom of the vessel. Dithering in all three dimensions may alsobe employed, as depicted in FIG. 3

For a relatively flat sample, such as whole leaf tissue, a histologicalsample, or thin-section specimen, where the area of the sample is largerelative to the cross-sectional area of the focal zone, the x-ypositioning system can cause the focal zone to traverse the sample inorder to treat the entire surface of the sample. This procedure may becombined with optical analysis or other sensors to determine the extentof the treatment to each portion of the sample that is brought into thefocal zone.

In certain embodiments, the sample or array of samples can be movedrelative to the transducer and the other parts of the apparatus. Inalternative embodiments the transducer is moved while the sample holderremains fixed, relative to the other parts of the apparatus. As analternative, movement along two of the axes, for example, x and y, canbe assigned to the sample holder and movement along the third axis, z inthis case, can be assigned to the transducer.

The three axis positioning system enables automated energy focusadjustment in the z axis when used in conjunction with a sensor formeasuring the ultrasound intensity. In one embodiment, a needlehydrophone can be mounted in a fixture on the sample positioning system.The hydrophone can be traversed in three dimensions through the focalregion to record the acoustic intensity as a function of position inorder to map out the focal zone. In another embodiment, a number ofpositions on a sheet of aluminum foil held in the sample holder can betreated in a sequence of z-axis settings. The foil can then be examinedto determine the spot size of the damage at each position. The diameterof the spot corresponds generally to the diameter of the focal zone atthat z-axis setting. Other, fully automated embodiments of a focusingsystem can also be constructed.

The three axis positioning system also allows the apparatus to beintegrated into a larger laboratory automation scheme. A positioningsystem with an extended work envelope can transfer microplates or othersample vessels into and out of the apparatus. This allows the apparatusto interact automatically up downstream processes.

D. Sensors

Visual Monitoring of the Sample

Optical or video detection and analysis can be employed to optimizetreatment of the sample. For example, in a suspension of biologicaltissue, the viscosity of the mixture can increase during treatment dueto the diminution of the particles by the treatment and/or by theliberation of macromolecules into the solution. Video analysis of thesample during treatment allows an automated assessment of the mixingcaused by the treatment protocol. The protocol may be modified duringthe treatment to promote greater mixing as a result of this assessment.The video data may be acquired and analyzed by the computer controlsystem that is controlling the treatment process. Other opticalmeasurements such as spectral excitation, absorption, fluorescence,emission, and spectral analysis also can be used to monitor treatment ofthe sample. A laser beam, for example, can be used for alignment and toindicate current sample position.

Monitoring of Temperature

Heating of individual wells can be determined by an infraredtemperature-sensing probe, collimated so as to view only the well beingtreated with the ultrasonic energy. For example, an infrared thermalmeasuring device can be directed at the top unwetted side of thetreatment vessel. This provides a non-contact means of analysis that isnot readily achievable in conventional ultrasound treatmentconfigurations. The thermal information can be recorded as a thermalrecord of the sample temperature profile during treatment.

Active temperature monitoring may be used as a feedback mechanism tomodify the treatment protocol during the treatment process to keep thesample temperature within specified limits. For example, an infraredsensor directed at the sample treatment vessel may input temperaturereadings to the computer. The computer, in accordance with a controllingprogram, can produce output directed to the circuit enabling theultrasonic transducer, which in turn can reduce the high power treatmentintervals and increase the low power mixing intervals, for example, ifthe sample temperature is nearing a specified maximum temperature.

Monitoring of Cavitation

A variety of methods may be employed to detect cavitation. For example,acoustic emissions, optical scattering, high-speed photography,mechanical damage, and sonochemicals can be used. As described above formonitoring temperature, information from cavitation detection can beused by the system to produce an output that selectively controlsexposure of a sample to sonic energy in response to the information.Each of these methods to monitor cavitation are described more fullybelow.

Acoustic emissions: Bubbles are effective scatterers of ultrasound. Thepulsation mode of a bubble is referred to as monopole source, which isan effective acoustic source. For small, generally linear oscillations,the bubble simply scatters the incident acoustic pulse. However, as theresponse becomes more nonlinear, it also starts to emit signals athigher harmonics. When driven harder, the bubbles start to generatesubharmonics as well. Eventually as the response becomes aperiodic orchaotic, the scattered field tends towards white noise. In the scenariowhere inertial collapses occur, short acoustic pressure pulses areemitted. An acoustic transducer can be configured to detect theseemissions. There is a detectable correlation between the onset of theemissions and cell disruption.

Optical scattering: Bubbles also scatter light. When bubbles arepresent, light is scattered. Light can normally be introduced into thesystem using fiber optic light sources so that cavitation can bedetected in real-time, and therefore can be controlled by electronic andcomputer systems.

High-speed photography: Bubbles can be photographed. This methodtypically requires high-speed cameras and high intensity lighting,because the bubbles respond on the time frame of the acoustics. It alsorequires good optical access to the sample under study. This method cangive detailed and accurate data and may be a consideration whendesigning systems according to the invention. Stroboscopic systems,which take images far less frequently, can often give similarqualitative performance more cheaply and easily than high-speedphotography.

Mechanical damage: Cavitation is known to create damage to mechanicalsystems. Pitting of metal foils is a particularly common effect, anddetection method. There is a correlation between the cavitation neededto pit foils and to disrupt cells.

Sonochemicals: A number of chemicals are known to be produced inresponse to cavitation. The yield of these chemicals can be used as ameasure of cavitational activity. A common technique is to monitor lightgeneration from chemicals, such as luminol, that generate light whenexposed to cavitation. Sonochemical yield usually can not be done duringcell experiments but can be done independently under identicalconditions, and thereby, provide a calibrated standard.

E. Temperature, Cavitation, and Pressure Management and Control.

Temperature Control

Certain applications require that the temperature of the sample beingprocessed be managed and controlled during processing. For example, manybiological samples should not be heated above 4° C. during treatment.Other applications require that the samples be maintained at a certainelevated temperature during treatment. The ultrasound treatment protocolinfluences the sample temperature in several ways: the sample absorbsacoustic energy and converts it to heat; the sample treatment vesselabsorbs acoustic energy and converts it to heat which, in turn, can heatthe sample; and acoustic streaming develops within the sample treatmentvessel and the water bath, forcing convective heat transfer between thesample treatment vessel and the water bath. In the case of a relativelycool water bath, this cools the sample.

The acoustic waves or pulses can be used to regulate the temperature ofthe solutions in the treatment vessel. At low power, the acoustic energyproduces a slow stirring without marked heating. Although energy isabsorbed to induce the stirring, heat is lost rapidly through the sidesof the treatment vessel, resulting in a negligible equilibriumtemperature increase in the sample. At higher energies, more energy isabsorbed, and the temperature rises. The degree of rise per unit energyinput can be influenced and/or controlled by several characteristics,including the degree of heat absorption by the sample or the treatmentvessel and the rate of heat transfer from the treatment vessel to thesurroundings. Additionally, the treatment protocol may alternate ahigh-powered treatment interval, in which the desired effects areobtained, with a low power mixing interval, in which acoustic streamingand convection are achieved without significant heat generation. Thisconvection may be used to promote efficient heat exchange or cooling.

The thermal information can also be used to modify or control thetreatment to maintain the sample temperature rise below a maximumallowable value. The treatment can be interrupted to allow the sample tocool down. In certain embodiments, the output of the thermal measurementdevice or system is entered into the computer control system forrecording, display on a control console, and/or control of exposure ofthe sample to sonic energy through a feedback loop, for example byaltering the duty cycle.

Temperature rise during ultrasonic continuous wave exposure can becontrolled, if required, by refrigeration of a liquid or other samplebefore, during, or after passage through a zone of sonic energy, ifprocessing in a continuous, flow-through mode. In generally stationarydiscrete sample processing modes, a sample can be cooled by air, bycontact with a liquid bath, or a combination of both air and liquid. Thetemperature is rapidly equilibrated within the vessel by the stirringaction induced by the acoustic waves. As a result, and especially insmall vessels or other small fluid samples, the rate of temperatureincrease and subsequent cooling can be very rapid. The rate of deliveryof sonic energy to the material can also be controlled, although thatcan lengthen processing time.

Liquids within the sample can be provided at any temperature compatiblewith the process. The liquid may be frozen or partially frozen forprocessing. For example, when biological material is subjected tosubzero temperatures below about −5° C., most, but not all, of the wateris in the solid phase. However, in certain biological tissues,micro-domains of liquid water still remain for several reasons, such asnatural “antifreeze” molecules or regions of higher salt concentration.Therefore, sample temperature may be varied during the procedure. Atemperature is selected at which microdomains of liquid water are ableto form shock wave induced cavitation due to bubble formation andcollapse, resulting in shear stresses that impinge on surroundingtissues. Indeed, gradually altering the sample temperature can bedesirable, as it provides focused domains of liquid water for collectionof sonic energy for impingement on the surrounding material.

Treatment baths can be relatively simple, and can include a water bathor other fluid bath that is employed to conduct the acoustic waves fromthe transducer to the sample treatment vessel, where the liquid istemperature controlled. In certain embodiments, the entire bath ismaintained at a specific temperature by means of an external heater orchiller, such as a Neslab RTE-210 chiller available from NeslabInstruments, Inc., located in Newington, N.H., and heat exchanger coilsimmersed in the bath. The sides and bottom of the tank containing thebath may have sufficient insulating properties to allow the bath to bemaintained substantially uniformly at a specific temperature. Anotherembodiment, such as that depicted in FIG. 9, employs an inner tray orsample tank 76 made of an insulating material such as rigid polystyrenefoam which is set within a larger water bath 84 in a transducer tank 82.The inner tray 76 has heat-exchanger tubes or other heating or coolingdevices within it (not shown) to allow a fluid 78 such as ethyleneglycol or propylene glycol in the inner tray 76 to be heated or cooledbeyond what may be practical for the fluid 84 such as water in the outerbath in the transducer tank 82. The inner tray 76 has an acoustic window88 in the bottom. The acoustic window 88 is made of a thin film materialhaving low acoustic absorption and an acoustic impedance similar towater. This inner tray 76 is arranged so that the acoustic window 88 isaligned with a transducer 86 which is outside the tray 76, supportedwith a support 80 in the water 84. A sample 74 is located within amicrotiter plate or other sample treatment vessel 72, within the tray 76and is subjected to the thermal influence of the inner treatment bath78. The treatment vessel 70 can be movable relative to the transducer 86with a positioning system 70. Also, sonic energy focuses on the sample74 through the acoustic window 88. This arrangement permits the use ofseparate fluids and substantially independent control of the temperatureof the inner 76 and outer treatment baths 84. The smaller volume of theinner tray 76 facilitates the use of antifreeze mixtures, such as amixture of propylene glycol and water, at temperatures below thefreezing temperature of water. This, in turn, allows the samples 74 tobe processed and treated at temperatures below the freezing temperatureof water. This embodiment is beneficial for treatment applicationsrequiring that the sample materials 74 be maintained at temperaturesnear or below the freezing point of water. It allows for the containmentof treatment bath fluids 78, such as antifreeze solutions, that may notbe compatible with the transducer 86 and other system components. Italso allows the transducer 86 to be maintained at a differenttemperature than the samples 74. This embodiment may also be connectedwith any of the other components described in FIG. 1 and is suitable foruse in a system with or without feedback loop control.

Sample temperature may be required to remain within a given temperaturerange during a treatment procedure. Temperature can be monitoredremotely by, for example, an infra-red sensor. Temperature probes suchas thermocouples may not be particularly well suited for allapplications because the sound beam may interact with the thermocoupleand generate an artificially high temperature in the vicinity of theprobe. Temperature can be monitored by the same computer that controlsacoustic waveform. The control responds to an error signal which is thedifference between the measured actual temperature of the sample and thetarget temperature of the sample. The control algorithm can be as ahysteritic bang-bang controller, such as those in kitchen stoves, where,as an output of the control system, the acoustic energy is turned offwhen the actual temperature exceeds a first target temperature andturned on when the actual temperature falls below a second targettemperature that is lower than the first target temperature. Morecomplicated controllers can be implemented. For example, rather thansimply turning the acoustic signal on and off, the acoustic signal couldcontinuously be modulated proportionally to the error signal, forexample, by varying the amplitude or the duty cycle, to provide finertemperature regulation.

In the application of a bang-bang control algorithm for a multiplesample format, once a maximum temperature value has been exceeded andthe sonic energy is turned off for a particular sample, an alternativeto waiting for the sample to cool below a selected temperature beforeturning the sonic energy on again, is to move on to the next sample.More particularly, some of the samples can be at least partially treatedwith sonic energy, in a sequence, and then, the system can return to thepreviously partially treated samples to take a sensor reading todetermine if the samples have cooled below the selected temperature andto reinitiate treatment if they have. This procedure treats the samplesin an efficient manner and reduces the total treatment time for treatingmultiple samples. Another alternative is to switch to a predefined“cooling” waveform which promotes convection without adding significantheat to a particular sample, rather than moving on to the next sampleand returning to the first sample at a later time.

If uniformity of temperature throughout the sample is important, thencontrol techniques can be used to ensure a uniform temperaturedistribution. An array of infra-red sensors can be used to determine thedistribution of the temperature inside the sample. If areas of increasedtemperature relative to the rest of the sample appear, then thetransducer can be switched from high power “treatment” mode to low power“mixing” mode. In the low power “mixing” mode, the sample isacoustically stirred until the sample is substantially uniform intemperature. Once temperature uniformity is achieved, the high power“treatment” mode is reinitiated. A control system can monitortemperature and responsively turn the various modes on or off. Whencontrolled by a computer, the intervals during which these modes areused can be very short, for example fractions of a second, thereby notsignificantly prolonging treatment times. Stepping times between wells,or other sample containers, can also be less than a second with suitabledesign.

Cavitation Control

In some applications, it can be preferable to treat the sample with asmuch energy as possible without causing cavitation. This result can beachieved by suppressing cavitation. Cavitation can be suppressed bypressurizing the treatment vessel above ambient, often known as“overpressure,” to the point at which no negative pressure developsduring the rarefaction phase of the acoustic wave. This suppression ofcavitation is beneficial in applications such as cell transformationwhere the desired effect is to open cellular membranes while maintainingviable cells. In other applications it may be desirable to enhancecavitation. In these applications, a “negative” overpressure or vacuumcan be applied to the region of the focal zone.

The control of cavitation in the sample also can be important duringacoustic treatment processes. In some scenarios, the presence of smallamounts of cavitation may be desirable to enhance biochemical processes;however, when large numbers of cavitation bubbles exist they can scattersound before it reaches the target, effectively shielding the sample.

Cavitation can be detected by a variety of methods, including acousticand optical methods. An example of acoustic detection is a passivecavitation detector (PCD) which includes an external transducer thatdetects acoustic emissions from cavitation bubbles. The signal from thePCD can be filtered, for example using a peak detector followed by a lowpass filter, and then input to a controlling computer as a measure ofcavitation activity. The acoustic signal could be adjusted in wayssimilar to those described in the temperature control example tomaintain cavitation activity at a desired level.

Overpressure: Increased ambient pressure is one technique forcontrolling cavitation. Overpressure tends to remove cavitation nuclei.Motes in the fluid are strongly affected by overpressure and socavitation in free-fluid is often dramatically reduced, even by theaddition of one atmosphere of overpressure. Nucleation sites oncontainer walls tend to be more resistant to overpressure; however thecavitation tends to be restricted to these sites and any gas bubblesthat float free into the free-fluid are quickly dissolved. Thereforecells in the bulk fluid are typically unaffected by cavitation sitesrestricted to the container walls. Overpressure may be applied to thetreatment vessel, the array of treatment vessels, the treatment bath andtank, or to the entire apparatus to achieve a higher than atmosphericpressure in the region of the focal zone.

Degassing: Reducing the gas content of the fluid tends to reducecavitation, again by reducing cavitation nuclei and making it harder toinitiate cavitation. Another method of controlling cavitation or theeffects of cavitation is to control the gasses that are dissolved in thesample fluid. For instance, cavitation causes less mechanical damage influid saturated with helium gas than in fluid saturated with argon gas.

Filtering: Cleaner fluids tend to be harder to cavitate.

Various fluids: Certain fluids are much harder to cavitate. Castor oiland mineral oil are nearly cavitation free. Two possible reasons arethat the fluids are of a nature that they tend to fill in cracks, andthat their viscosity also makes them more resistant to cavitation. Thefluids, however, are not particularly compatible with cell preparations.

Waveform shape: The cavitation field responds to the acoustic drivingpulse. It is possible to control the cavitation response, to someextent, by controlling the driving acoustic pressure. Cavitation mayalso be reduced or eliminated by reducing the number of cycles in eachburst of acoustic energy. The cavitation bubbles grow over severalcycles then collapse creating cavitation effects. By limiting the numberof cycles in each burst, bubble growth and collapse can be substantiallyavoided.

F. Treatment or Reaction Vessel

Treatment vessels are sized and shaped as appropriate for the materialto be treated. They can be any of a variety of shapes. For example, asshown in FIGS. 4A-4C, treatment vessels 502, 504, 506 can have verticalwalls, can have a conical shape, or can have a curved shape,respectively. As shown in FIGS. 5A-5C, certain treatment vessel 502,506, prior to treatment with sonic energy, have an upper member 530 anda lower member 550 which together form an interior region that containsthe material 540 to be treated. In certain embodiments, the ultrasoundtransducer projects a focused ultrasound beam upwards. The ultrasoundbeam penetrates the lower member 550 of the treatment vessel 502, 506 toact upon the contents 540 of the treatment vessel 502, 506. The uppermember 530 serves to contain the contents 540 of the vessel 502, 506.

The lower member 550 of the treatment vessel 502, 506 is configured totransmit the maximum amount of ultrasound energy to the contents 540 ofthe vessel 502, 506, minimize the absorption of ultrasound energy withinthe walls of the vessel 502, 506 and maximize heat transfer between thecontents 540 of the treatment vessel 502, 506 and, for example, anexternal water bath. In certain embodiment of the pre-treatmentassembly, the treatment vessel is thermoformed from a thin film in ahemispherical shape. The film should have an acoustic impedance similarto that of water and low acoustic absorption. One preferred material islow density polyethylene. Alternative materials include polypropylene,polystyrene, poly(ethylene teraphthalate) (“PET”), and other rigid andflexible polymers. The film may be a laminate to facilitate thermalbonding, for example using heat sealing. Thicker, more rigid materialsmay also be employed. Available multi-well plates in industry standardformats such as 96 well and 24 well formats may be employed with orwithout modification. Industry standard thick-wall, multi-well plateswith thin film bottoms may also be employed. These can work particularlyadvantageously where the size of the focal zone of the ultrasound beamis smaller than a well. In this case, little energy is absorbed by thesides of the treatment vessel and, as a result, relatively little energyis converted to heat.

The upper member of the treatment vessel contains the contents in thevessel during treatment and can act also as an environmental seal. Theupper member of the treatment vessel can be flat or domed to enclose theinterior of the treatment vessel. The upper member of the treatmentvessel may be made of a rigid or flexible material. Preferably, thematerial will have low acoustic absorption and good heat transferproperties. In certain embodiments of the pre-treatment assembly, theupper member of the treatment vessel is a thin film that can be bondedto the lower member, and the lower or upper member can be easilyrupturable for post-treatment transfer of the treated material.

The upper and lower members of the treatment vessel may be joinedtogether by thermal bonding, adhesive bonding, or external clamping.Such joining of the upper and lower members can serve to seal thecontents of the vessel from contaminants in the external environmentand, in an array of vessels, prevent cross-contamination betweenvessels. If the bond is to be achieved by thermal bonding, the upper andlower members of the treatment vessels may be made of film laminateshaving heat bondable outer layers and heat resistant inner layers.

The treatment vessel may be configured as a single unit, as amultiplicity of vessels in an array, or as a single unit with variouscompartments. The upper and lower members of the vessel or array ofvessels can be used once or repeatedly. There also can be a separateframe or structure (not shown) that supports and/or stiffens the upperand lower members of the vessel(s). This frame or structure may beintegral with the vessels or may be a separate member. An array oftreatment vessels may be configured to match industry standardmulti-well plates. In one embodiment the treatment vessel is configuredin an array that matches standard 96 well or 24 well multi-well plates.The frame or supporting structure holding the array of treatment vesselscan have the same configuration and dimensions as standard multi-wellplates.

As shown in FIGS. 6A and 6B, a treatment vessel 508 can include a funnel592 to facilitate transfer of the contents 540 from the treatment vessel508 to a separate vessel 598 after treatment. The funnel 592 can have aconical shape and include an opening at the narrow end. The funnel 592can be rigid, relative to the upper 530 and lower members 550 of thetreatment vessel 508. The large end of the funnel 592 is proximate theupper member 550 of the treatment vessel 508 and aligned with thetreatment vessel 508. The volume of the funnel 592 can be marginallyless than the volume of the treatment vessel 508.

One process of transferring the contents 540 of the treatment vessel 508to another post-treatment vessel 598 includes the following steps. Theupper member 530 of the treatment vessel 508 may be pierced with a sharpinstrument or ruptured when a vacuum is applied. To facilitate rupture,the member 530 may be manufactured from a thin fragile material or madeweak by etching a feature into the surface. Then, the treatment vessel508 is inverted over the post-treatment vessel 598 in a vacuum fixture.A filter 594 may be placed between the treatment vessel 508 and thepost-treatment vessel 598 to separate solids 596 from the liquid 542that is removed from the treatment vessel 508. Alternatively, the filter594 may be incorporated into the outlet of the funnel 592. Thisarrangement of treatment vessel 508 and funnel 592 may be configured asa single unit or as an array of units. This array may match an industrystandard. The treatment vessel 508 should form a vacuum seal with avacuum fixture (not shown) such that a pressure differential can formbetween the sample in the treatment vessel and the supplied vacuum. Oncethe vacuum is applied to the fixture, the pressure differential acrossthe upper member 530 will cause the upper member 530 of the treatmentvessel 508 to rupture and cause the lower member 550 to collapse intothe funnel 592. The lower member 550 should have sufficient strength sothat it does not rupture where it bridges the opening in the small endof the funnel 592. The pressure differential will cause the solidcontents 596 of the treatment vessel to be squeezed between the flexiblelower member 550 of the treatment vessel 508 and the relatively rigidfunnel 592. This causes fluid 542 to be expelled from the solidmaterials 596 and collected in the post-treatment vessel 598.

In certain other embodiments, a treatment vessel can be an ampoule,vial, pouch, bag, or envelope. These and other treatment vessels can beformed from such materials as polyethylene, polypropylene, poly(ethyleneteraphthalate) (PET), polystyrene, acetal, silicone, polyvinyl chloride(PVC), phenolic, glasses and other inorganic materials, metals such asaluminum and magnesium, and laminates such as polyethylene/aluminum andpolyethylene/polyester. Also, certain embodiments of a treatment vesselcan be made by vacuum forming, injection molding, casting, and otherthermal and non-thermal processes. In embodiments where samples flowthrough the sonic energy, capillary tubes, etched channels, and conduitsmay be the sample holder during treatment as the sample flows through astructure. Additionally, free-falling drops, streams, non-moving freevolumes, such as those in gravity less than one g, or a layer in adensity gradient can be treated directly.

II. Materials for Treatment

A. Biological Materials

Many biological materials can be treated according the presentinvention. For example, such materials for treatment include, withoutlimitation, growing plant tissue such as root tips, meristem, andcallus, bone, yeast and other microorganisms with tough cell walls;bacterial cells and/or cultures on agar plates or in growth media, stemor blood cells, hybridomas and other cells from immortalized cell lines,and embryos. Additionally, other biological materials, such as serum andprotein preparations, can be treated with the processes of theinvention, including sterilization.

B. Binding Materials

Many binding reactions can be enhanced with treatments according to theinvention. Binding reactions involve binding together two or moremolecules, for example, two nucleic acid molecules, by hybridization orother non-covalent binding. Binding reactions are found, for example, inan assay to detect binding, such as a specific staining reaction, in areaction such as the polymerase chain reaction where one nucleotidemolecule is a primer and the other is a substrate molecule to bereplicated, or in a binding interaction involving an antibody and themolecule it binds, such as an immunoassay. Reactions also can involvebinding of a substrate and a ligand. For example, a substrate such as anantibody or receptor can be immobilized on a support surface, for use inpurification or separation techniques of epitopes, ligands, and othermolecules.

C. Chemical and Mineral Materials

Organic and inorganic materials can be treated with controlled acousticpulses according to the methods of the invention. The sonic pulses maybe used to comminute a solid material, particularly under a feedbackcontrol regime, or in arrays of multiple samples. As with biologicalsamples, individual organic and inorganic samples in an array can betreated in substantial isolation from the laboratory environment. Besidealtering their physical integrity, materials can be dissolved in solventfluids, such as liquids and gasses, or extracted with solvents. Forexample, dissolution of polymers in solvents can be very slow withoutstirring, but stirring multiple samples with current methods isdifficult and raises the possibility of cross-contamination betweensamples. However, stirring of multiple samples withoutcross-contamination between samples can be accomplished with apparatusand methods of the present invention

III. Treatment Applications

A. Altering Cell Accessibility

Sonicators can disrupt cells using frequencies around 20 kHz. It isgenerally thought there are two ways in which ultrasound can affectcells, namely by heating and by cavitation, which is the interaction ofthe sound wave with small gas bubbles in the sample. Heating occursprimarily due to absorption of the sound energy by the medium or by thecontainer. For dilute aqueous systems, it is absorption by the containerthat is a main source of the heating. Heating is not desirable in sometreatment applications, as described herein. The heating associated withthe compression and cooling associated with the rarefaction of a soundwave is relatively small, even for intense sound.

According to the invention, controlled sonic pulses in a medium are usedto treat a sample containing biological material. The pulses can bespecifically adapted to preferentially interact with supporting matricesin a biological material, such as plant cell walls or extracellularmatrices such as bone or collagen, thereby lessening or removing abarrier function of such matrices and facilitating the insertion ofextracellular components into a cell. In this application, the cell isminimally altered and cell viability is preserved. These pulses can becaused by shock waves or by sound waves. The waves can be createdexternal to the sample, or directly in the sample, via appliedmechanical devices. In experiments where thermal effects are negligible,there typically is no lysis, unless cavitation is present. Other modesof sonic energy can have different effects than disrupting a matrix andcan be used either with pre-treatment, with disrupting sonic energy, orby themselves. For, example the conditions to disrupt a matrix can bedifferent from those to permeabilize a cell membrane.

There are many possible mechanisms by which cavitation may affect cellsand there is no consensus as to which mechanisms, if any, dominate. Theprinciple mechanisms are thought to include shear, microjets, shockwaves, sonochemistry, and other mechanisms, as discussed more fullybelow.

Shear: Significant shear forces are associated with the violent collapseof bubbles. Because cell membranes are sensitive to shear, it is thoughtthat cavitation may permeabilize cell membranes. In some cases, themembrane is apparently permeable for only a short time, during whichmolecules may be passed into or out of the cell. In other cases the cellmay be lysed.

Microjets: Bubbles undergoing a violent collapse, particularly near aboundary, such as a container wall, typically collapse asymmetricallyand generate a liquid jet of fluid that passes through the bubble andinto the boundary. The speed of this jet has been measured to behundreds of meters a second and is of great destructive power. It mayplay a major role in the destruction of kidney stones by acoustic shockwaves and may be a possible way of destroying blood clots.

Shock wave: Collapse of a bubble spherically can generate an intenseshock wave. This pressure can be thousands of atmospheres in theneighborhood of the bubble. The compressive stress of the shock wave maybe strong enough to cause cellular material to fail.

Sonochemistry: The pressure and temperatures in the bubble during aninertial collapse can be extraordinarily high. In extreme examples, thegas can be excited sufficiently to produce light, termedsonoluminescence. Although the volume is small and the time durationshort, this phenomenon has been exploited to enhance chemical reactionrates. The production of free-radicals and other sonochemicals may alsoaffect cells.

Other: Other factors also may be involved. Vessel walls may contributecavitation nuclei. A plastic vessel with an aqueous fluid may result ina standing wave field due to internal reflections, as a result ofimpedance mismatches between the fluid and the vessel walls. Examples ofsonolucent materials are thin latex and dialysis tubing. Tube rotationstudies performed on continuous wave dosage with unfocused ultrasonicsindicate that rotation has a significant effect on hemolysis. When cellcontents were mechanically stirred during insonation, the cell lysisincreased. These effects may be due to viscosity gradients set-up withinthe unfocused ultrasound field that block energy transmission.

Cellular lysis also can be aided by the addition of ultrasound contrastagents, such as air-based contrast agents or perfluorocarbon-basedcontrast agents. An example of an air-based contrast agent is adenatured albumin shell with air such as Albunex, available fromMallinckrodt, St. Louis, Mo., and an example of a perfluorocarbon-basedcontrast agent is a phospholipid coating with perfluoropropane gas suchas MRX-130, available from ImaRx Pharmaceutical Corp., Tucson, Ariz.

Air bubbles can block or reflect energy transmission. Interfaces betweenair and water result in efficient reflection of an incident ultrasoundfield.

The treatment dose is a complex waveform. Sections, or components, ofthe waveforms can have different functions. For example, the waveformcan have three components involved with sample mixing, samplelysis/disruption, and sample cooling.

In other current methods, sonolytic yield activity decreases withincreasing cell concentrations in in vitro systems that are treated withcontinuous ultrasound waves. In contrast, methods according to thepresent invention disrupt tissue structures with a complex waveform ofhigh intensity focused ultrasound, to avoid this problem.

Mixing can be an important, because it allows bubbles that may have beendriven by radiation forces to the edges of the vessel chamber to bebrought into contact with the cell or tissue membranes. This mixingpromotes inertial, transient acoustic cavitation near the cell walls,resulting in cellular lysis.

The acoustic dosage received by a sample can be likened to a radiationdosage received by a sample. In each case, a cumulative effect of theabsorbed energy dose is observed. A computer-controlled positioningsystem can control the cumulative energy dosage that each samplereceives. For example, a software program in the computer can activelycontrol the cumulative energy dosage by treating the sample until thesystem reaches a particular set-point, pausing energy application orotherwise allowing the sample to reequilibrate, and reinitiating energyapplication to allow a sample to receive a higher cumulative dose whilemaintaining semi-isothermal conditions, such as a 1 to 2 degreeCentigrade temperature rise during exposure, than would otherwise bepossible by continuous sonic energy application. This type of systemenables high energy to be introduced into a sample while maintainingthermal control of the process.

B. Extracting

In a variation of the method to alter cellular accessibility describedabove, controlled pulses in a medium can be used to treat a samplecontaining biological material to extract a fraction or fractions of thebiological material. The pulses are specifically adapted topreferentially interact with supporting matrices, such as plant cellwalls or extracellular matrices such as bone or collagen, or materialshaving differences in rigidity or permeability in a biological material,thereby lessening or removing a barrier function of such matrices ormaterials. These pulses can be caused by shock waves or by sound waves.The waves can be created external to the sample, or directly in thesample, via applied mechanical means.

Using sound energy, as opposed to laser or other light energy to disrupta biological object, can be useful. Sound is a direct fluctuation ofpressure on the sample. Pressure is a physical quantity and the measureof uniform stress defined as the force per unit area. The stress actingon a material induces strain which changes dimensions of the material.The two main types of stress are a direct tensile or compressive stressand shear stress. In general, the more brittle the material, the greaterthe disruptive effect of an abrupt, local increase of otherwise uniformstress. Such a local stress can be created by some geometric change at asurface or within the body of the sample. For example, biological tissuefrozen at −70° C. may be more prone to stress fracture than at 4° C. Inaddition, a sharper change in geometric or material properties tends tocause a greater stress concentration, which in turn can yield a greaterdisruption. Sound waves may be focused. In contrast, the energytransferred from a light source such as a laser to a sample iselectromagnetic radiation that induces non-ionizing molecular vibrationsand breaks chemical bonds by ionizing. Mechanical stress on objectslarger than molecules generally cannot be readily caused byelectromagnetic waves, except via destructive local heating.

The supporting matrix of a biological sample can be disrupted withoutdisrupting one or more selected internal structures of the cellscontained within the matrix. Representative examples of such samplesare: i) bone, in which a rigid matrix contains living cells of interest;ii) mammalian tissue samples, which contain living cells embedded in amatrix of elastic connective tissue and “glycocalyx” or intercellularmatrix; and iii) plant tissues, such as leaves, which contain cells in amatrix of cellulose, often crosslinked with other materials, of moderaterigidity. Virtually all living cells are gelatinous in texture, and canbe deformed to some extent without rupture or internal damage. Matrices,in contrast, are designed to support and protect cells, as well as toachieve other biological functions. In the three examples above, thematrices of bone and leaves are designed to provide rigidity to thestructure, while the support of most collagenous matrices has a stronglyelastic character. Thus, different protocols for example, amplitude,duration, number of pulses, and temperature of sample, may be used todisrupt different matrices by mechanical means without damaging thecellular material.

A bony matrix is both more rigid and denser than the cells it contains.Bone is vulnerable to shock waves, both because the calcified matrixwill absorb the waves more efficiently than will the cells, and becausethe calcified matrix is weak under extensional strain, and thereby canfragment at stresses which will not damage the softer cells. Similarconsiderations apply to leaf matrix, although the contrast in densityand modulus is less. In either case, a pulse, preferably a shock wave,is applied at an amplitude which is sufficient to fatigue the matrixcomponents while remaining below the amplitude required to damage thecells. This intensity is determined readily for a particular type ofsample by minimal routine experimentation. In such experiments, theamplitude of each pulse applied to the sample, singly or in a train ofpulses, is varied to obtain the maximum rate of degradation of thematrix consistent with retention of the viability of the cells withinthe matrix. These parameters can be measured readily. For example,matrix degradation can be measured by variation in the compressivemodulus of the sample, while cell integrity is measured by dye exclusionfrom cells extracted from the matrix, such as, for bone,demineralization and treatment with collagenase. In the case of a moreelastic tissue, such as connective tissue, which is cross-linked but hasa high extension to break, the pulses are selected to excitepreferentially vibrational modes in the matrix in contrast to the cells.This can be done by selecting one or more frequencies of sound waves atwhich the relative absorptiveness of the matrix and the cells aremaximally different. Such frequencies are determined readily by routineexperimentation. A sequence of pulses may be required to differentiallyfatigue the matrix. The length of the pulses and the interval betweenthem are adjusted so that the degree of heating of the sample does notcause loss of integrity of the cells, and particularly of the criticalcomponents which are to be isolated.

Three areas to optimize for extraction are treatment waveform, mixingwaveform, and positioning or dithering. One method to determine theappropriate treatment and positioning parameters for a target sample forextraction purposes is described below.

First, a solid sample is placed in a volume of liquid in about a 1:1ratio (weight/volume), in a treatment vessel. For example, 0.25 ml ofmethanol is added to 0.25 gm of leaf tissue in a 0.5 ml treatmentvessel. A single sample is placed within the focal zone of the sonicapparatus. Without using the treatment protocol, the mixing waveform isadjusted to provide “stirring” of the sample at the lowest amplitude,fewest cycles/burst, and lowest duty cycle. After the mixing waveformprotocol is defined, the disruption treatment waveform is adjusted byimmobilizing the target sample in the focal zone such that there is nomixing and no sample movement, such as dithering. Using a sonic energysource such as a piezoelectric transducer, the sample is subjected to aminimum number of cycles per burst, for example, three. For extractionpurposes, the amplitude is initially used with a nominal 500 mV setting.A portion of the sample is treated and inspected under a microscope forsigns of membrane disruption. Such inspection can be done in conjunctionwith dyes that stain intracellular organelles. The number ofcycles/burst is then increased until a particular desired tissuedisruption level is achieved in the immobilized portion of tissue. Witha fresh sample, and with a 1:1 ratio of tissue to liquid, thetemperature of the sample is monitored during a million cycle totaltreatment with an infra-red sensor directed to the top of a thinpolyethylene film covering the sample vessel. The duty cycle is adjustedto keep the temperature within predefined ranges, such as 4° C. within±2° C.

Once these treatment parameters are discerned for a particular sample, acontrol unit can be programmed with these data in order to controltreatment of other samples of the same or similar biological type.Alternatively, such information can preprogrammed in the control unit,and an apparatus user, through a user input interface, can designate thebiological material type to be treated such that the controller thenruns through the predetermined treatment cycle. Other information can beempirically determined for optimal treatment of a particular biologicalmaterial in a manner similar to that described above. For example,parameters such as treatment waveforms, mixing waveforms, and samplepositioning can be discerned. These parameters can vary depending uponthe particular biological material, the particular liquid that surroundsthe sample, and/or the particular treatment vessel used duringtreatment.

C. Introducing a Molecule into or Removing a Molecule from a Cell

Once a sample having a matrix has been sufficiently weakened orattenuated, but not to the point where a substantial number of cellscontained within the matrix are killed or lysed, an exposed target cellor cells become amenable to insertion of exogenous molecules bytechniques such as transfection or transformation. With some matrices,it may be convenient to isolate the cells from the matrices and then totransfect the cells. In other cases, it will be preferable, particularlyin an automated system, to perform the transfection directly on thetreated tissue sample, using solutions and conditions adapted from knowntechniques. Alternatively, in situations where a cell to be treated isnot situated within a matrix, the cell can be directly treated accordingto the process below without having to pre-treat the matrix. While thetreatment below is described mainly for transfection, methods andapparatus according to the present invention are equally applicable to atransformation process or other processes to introduce an exogenousmaterial into a permeabilized cell membrane.

In general, cool temperatures, less than 25° C., preferably less than15° C., more preferably 4° C. or below, tend to minimize the degradativeeffects of enzymes in the sample and thereby tend to preserve theintegrity of biological components to be isolated. However, cells,especially mammalian cells, may maintain their viability better athigher temperatures, such as 30 to 37° C. These temperatures also allowenzymes to be added to aid in the selective destruction of the matrix.

Alternatively, the sample temperature may be below 0° C. Except underspecial conditions, this will freeze the sample, or maintain it in afrozen state. Freezing can be advantageous if it enhances the disruptionof the matrix while allowing the cell to remain relatively intact. Forexample, ice crystals formed on freezing can be selectively largeroutside of cells. Since such crystals may tend to absorb acousticalenergy better than water, destruction of the matrix may be enhanced.While decreasing cell viability and integrity, such a procedure couldenhance the ease of transfection with exogenous material after thawingof the sample.

The waveforms used to alter the permeability of a cell are refineddepending on the particular application. Typically, the shock wave ischaracterized by a rapid shock front with a positive peak pressure, forexample about 100 MPa, and a negative peak pressure, for example aboutnegative 10 MPa. This waveform is of a few microsecond duration, on theorder of about 5 microseconds. If the negative peak is greater thanabout 1 MPa, cavitation bubbles may form. Cavitation bubble formation isalso dependent upon the surrounding medium. For example, glycerol is acavitation inhibitive medium; whereas, liquid water is a cavitationpromotive medium. The collapse of cavitation bubbles forms “microjets”and turbulence that impinge on the surrounding material.

Sound waves, namely acoustic waves at intensities below the shockthreshold, provide an alternative means of disrupting the matrix toallow access to the plasma membranes of the cells to allowtransformation. Such sound waves can be generated by any known process.As biological material is subjected to subzero temperatures, for exampleabout negative 5° C., most but not all of the water is in the solidphase. However, in certain biological tissues micro-domains of liquidwater still remain for several reasons, such as natural “antifreeze”molecules or regions of higher salt concentration. Therefore, as asample temperature is varied during the treatment with sound or shockwaves, microdomains of liquid water are able to form shock waves andinduce cavitation bubble formation and collapse, with the resultantshear stresses that impinge on surrounding tissues. Indeed, gradualalteration of the sample temperature can be desirable, as it providesfocused domains of liquid water for impingement on the surroundingmaterial. The waves can be applied to the samples either directly, aspiezoelectric pulses, or via an intervening medium. This medium may bewater, buffer, stabilizing medium for the target material to beisolated, or extraction medium for the target. An intervening mediumalso can be a solid, formed of a material which is intrinsically solid,or of a frozen solution. Waves also can be applied through a container,such as a microtiter plate.

The techniques useful for disrupting matrix structure can be adapted,and the improved technique can be used, to facilitate the incorporationof exogenous material into cells. The exogenous material may be DNA,RNA, other nucleic acid constructs, nucleic acid monomers, plasmids,vectors, viruses, saccharides, polysaccharides, amino acids, amino acidchains, enzymes, polymers, organic molecules, inorganic molecules,proteins, cofactors, and/or visualization reagents such as fluorescentprobes. In this application, shock waves or sonic waves are used toloosen the matrix, essentially as described above. However, theintensity of application of acoustic energy is kept sufficiently short,or below a critical energy threshold, so that cell integrity iscompletely maintained, as verified by a method such as dye exclusion.

At that point, or, optionally, previously, a solution or suspensioncontaining the material to be incorporated into the cells is added tothe sample. In one embodiment, the exogenous material is incorporatedinto the cells in a conventional manner, as is known in the art forcells with exposed plasma membranes. In another embodiment, acousticenergy is used to transiently permeabilize a plasma membrane tofacilitate introduction of exogenous materials into the cells. Theexogenous material may be present in the sample during the weakening ofthe matrix by acoustic energy. Even when the cells remain intact, asdetermined by dye exclusion or other viability measurements, the processof weakening the cell matrix by acoustic energy transiently destabilizesthe plasma membranes, increasing the uptake of exogenous macromoleculesand structures. If a further increase in the rate of incorporation isneeded, then the intensity or time of application of acoustic energy isslightly increased until the cell membrane becomes transientlypermeable. For example, a gentle pulse or wave is applied to themixture, with a predetermined amplitude. This amplitude can bedetermined readily in separate experiments on samples of the same typeto transiently make a plasma membrane of a cell type porous, in asimilar empirical manner to the steps described above for determining anappropriate treatment to disrupt a matrix. During the transient porousstate, exogenous materials diffuse into the cell and the materials aretrapped there once the sonic or shock pulse is removed.

A major advantage of these methods for transfection, or otherincorporation of exogenous material into living cells, is that themethods are readily amenable to scale-up, to automation, and to markedreduction in sample size and reagent volume. The wells of microplatescan be used for sonic treatment, transfection, and post-transfectiondemonstration of successful incorporation of the added material. Forexample, extracellular non-incorporated reagent, for example afluorescent material, can be inactivated by a material that does notpass the cell membrane, such as an enzyme, or certain hydrophilic oramphiphilic small-molecule reagents. Then the presence or absence of therequired material can be determined directly in the sample, for exampleby spectroscopy. Thus, the methods are adaptable to large scaleautomation, in large part because they do not require the isolation ofthe cells from their matrix. Additionally, these methods are amenable toa continuous flow process such as that described for sterilization,below. For example, the sonic energy treatment can be different forpermeabilization than for sterilization, but the sample to be treatedcan be flowed through an apparatus similar to that described forsterilization in FIG. 7.

The permeabilized cells can be transformed or transfected, usingtechniques known to those skilled in the art, for example,electroporation, vacuum transfection, or using viral vectors,agrobacterium, liposomes or other delivery vehicles, plasmids, or nakednucleic acids. The buffer conditions may be altered during the process.For example, the initial permeabilization may occur with chemicals toselectively alter the external cell wall, while during the nuclear wallpermeabilization step, other chemicals or biochemicals may be added toprompt selective uptake.

Additionally, with the process of permeabilization and with the mixingprofile, other techniques of gene transfer may be augmented. Examplesinclude, calcium phosphate coprecipitation, electroporation, andreceptor-dependent processes.

D. Sterilizing

The terms “sterilize,” “disinfect,” “preserve,” decontaminate,”“inactivation,” “disinfect,” and “kill” are used interchangeably herein,unless otherwise demanded by the context. “Sterilization,” namelykilling of all organisms, may not be synonymous in certain operationswith “decontamination,” for example, when the contaminant is non-living,such as a protein or prion. These terms, typically, mean the substantialelimination of or interference with any activity of a particularorganism and/or particle.

Methods for permeabilization and extraction, described above, can bemodified to sterilize a sample. The apparatus and methods forsterilizing can be optimized for efficient sterilization of particularmaterials in particular volumes and containers. For a particularmaterial to be sterilized, an initial set of conditions is selected.Such conditions can include selection of a type of sonic pulsegenerator, intensity of sonic energy, frequency of sonic energy, whererelevant, and/or like variables. The conditions also can include volume,mode of transport, and/or exposure of the materials to be sterilized.Then, the initial conditions and near variants are applied to thesample, and the percentage of cells or viruses killed is determined bystandard assay conditions. Further variables are selected for change.Accordingly, a zone of maximal killing of the test organism is found.Finally, other variables, such as flow rate and/or length and/orintensity of sonic exposure, are optimized to provide both a technicalsolution and a commercially useful solution to the problem ofsterilizing a particular material. Any of these empirically determinedvalues can be programmed into a control system of an apparatus used forsterilization to actively control sterilization, or the apparatus canhave these values previously determined such that a user need onlyselect a predetermined sterilization mode an the apparatus.

For many liquids, adequate sterilization is provided by destroying thecell walls of bacteria, fungi, and other living cells. This result isaccomplished by using frequencies and wavelengths of sound whichpreferentially excite the membranes of the cells while minimally heatingthe solution until the cells are lysed. In most cellular organisms,opening the membrane and allowing the contents to mix with anextracellular fluid will kill the organism.

Viruses can be opened to the solution by similar processing. In the caseof viruses, exposure of their internal nucleic acid to the solution maynot be adequate to completely inactivate them, since the naked DNA orRNA can also be infectious. Adjuncts such as iodine or nucleic-aciddigesting enzymes in the solution can be provided to complete theinactivation of the viruses.

Now, referring to FIG. 7, a schematic illustration for an apparatus 50to sterilize a continuous flow fluid is shown. For example, but withoutlimitation, the apparatus can be used to sterilize blood or other fluidssupplied to a patient. In this embodiment, fluid flows through the lumenof a conduit 54 between a first connector 62 and a second connector 56.The connectors 56, 62 can be Luer fittings and the connectors can beconnected with other tubing and/or devices (not shown) that provide orreceive the fluid. The fluid moves between the connectors 56, 62 in adirection indicated by an arrow 58. A sonic energy source 60, such as ahigh intensity focused ultrasound transducer, is located adjacent theconduit 54 and the sonic energy is emitted to a focal zone at leastpartially within the conduit 54. Many different arrangements of a sonicenergy source or sources are possible, such that sonic energy is emittedto a focal zone into the fluid contained within the conduit. Thetemperature of the fluid flowing through the conduit 54 can be monitoredwith a sensor (not shown) that, for example, receives infrared energyfrom the fluid as it flows through the conduit 54. Alternatively, theconduit can have at least one window or thin membrane portion whichallows infrared radiation to pass through to the sensor. A computer withan adaptive control can provide precise and accurate control of thetemperature of the medical fluid during the treatment in a mannersimilar to that described above. Also, during the ultrasonic treatment,feedback control can stabilize the temperature at a desired value, in amanner similar to that described above, to maintain the integrity and/orviability of fragile components within the fluid. For example, if thefluid is blood, one fragile component that is maintained can be FactorVIII. In addition, flowing the fluid past the focal point maintains a“bubble-free” focal zone. While blood might be removed from a patient,treated according to the invention outside the patient, that is treatedex vivo, and returned to the patient, other treatment situations arepossible. For example, one person's blood can be removed and treatedaccording to the invention and then given to a second person during atransfusion. Additionally, the sterilizing qualities of treatmentsaccording to the invention are contemplated to be useful whenever afluid needs to be sterilized.

In another sterilizing treatment mode and apparatus, and especially forhigh-volume applications, a wide, shallow zone of sterilizing sonicenergy can be created by apposition of a pair of plates to form asterilizing cell. At least one of the plates is an emitter of sonicenergy. The sterilizing cell is sealed appropriately such that the cellhas a sealed inner volume, with connections for fluid flow into and outof the cell. Fluid flow through the cell can be substantially laminarunder these circumstances, facilitating proper flow rate selection toprovide sufficient exposure of the fluid to the sonic energy to producesterilization.

In an alternative sterilizing treatment mode and apparatus, fluid isconveyed through a zone of sterilizing sonic energy by being pumpedthrough the zone in a conduit. The conduit itself may be immersed in aliquid or solid material that is designed to improve the efficiency withwhich sonic energy from the sonic energy emitter is provided to theconveyed fluid. The conduit also can be directly connected to a sourceof sonic energy, such as a tubular piezoelectric wave source. Ifchemical compatibility is adequate, a portion of the conduit itself maybe made of a material that can generate the sonic waves, such as apiezoelectric ceramic. Alternatively, any of these treatment processesmay be a manufacturing batch process for intravenous products.

E. Mixing, Stirring, and Heating

In fluid samples, including powdered and granular media and gasses,sample mixing is conventionally performed by vortexing or stirring, orother methods such as inversion of a sample containing an air space, andshaking. Vortexing is essentially achieved by mechanical motion of theentire vessel while stirring involves mechanical contact of a drivendevice with a fluid. Stirring is accomplished with a variety of devices,for example with propellers, impellers, paddles, and magnetic stir bars.One problem with these methods is that it is difficult to increase theirscale in order to handle dozens or hundreds of sample vessels at once.Another problem with these methods is the difficulty of mixing multiplesamples while keeping the each sample substantially free fromcontamination. As described in more detail below, methods according tothe invention can use sonic energy to mix a sample while avoidingproblems with contamination. Factors, such as focusing the sonic energy,as well as otherwise controlling an acoustic waveform of the sonicenergy, can be used to selectively mix a sample, for example, throughacoustic streaming and/or microstreaming.

A fluid sample can be mixed controllably using the system describedherein. No direct contact between the material to be mixed and the sonicenergy source is required. When the material to be mixed is in atreatment vessel, such as a microplate, the treatment vessel itself isnot necessarily touched by the source and is typically coupled to thesource by a fluid bath.

In certain embodiments, a treatment process for sample mixing in atreatment vessel can be summarized as follows. First, a sample istreated with sonic energy at a relatively high first treatment power inorder to heat the sample by absorption of acoustic energy. Second, thesample is mixed at a second sonic energy power, which may be the same orlower than the first treatment power, to cool the sample back to itsoriginal temperature by forcing convection through material in thetreatment vessel, which can be in contact with a fixed-temperature bathor reservoir.

In some embodiments, a source of focused ultrasonic waves is used. Thesource is mounted in a water bath or equivalent, which can providetemperature control. The microplate, with samples in the wells, ispositioned so that the focus of the beam is within the well. The plateis positioned so that the bottoms of the wells are in contact with orimmersed in the water or other fluid in the bath. Then, a burst ofultrasonic energy is applied to the well. This burst will cause stirringin the well, by formation of a convection cell. The stirring is easilyvisualized by adding particulate material to the wells, or by adding adye in a denser or lighter solution.

It is possible to select a sound field which will stir all of the wellsof a plate at one time. In one embodiment, a substantially uniform fieldis projected to the plate by a source, which preferentially excites thebottoms of the wells. This excitation in turns drives convective flow ineach of the wells.

In any embodiment, it can be useful to move the sample treatment vessel,such as by “dithering” the plate or well being treated relative to thesource. Dithering, as used in optics and in laser printing, is a rapidside to side two or three dimensional movement of the energy sourceand/or the target. Dithering, or other types of motion, can even outvariations in source intensity due to variations in the emitted sonicenergy or the location of the sample with respect to the source.Dithering can also prevent particulates from accumulating at the wall ofthe well.

F. Enhancing Reactions and Separations

In certain embodiments, temperature, mixing, or both can be controlledwith ultrasonic energy to enhance a chemical reaction. For example, theassociation rate between a ligand present in a sample to be treated andan exogenously supplied binding partner can be accelerated. In anotherexample, an assay is performed where temperature is maintained andmixing is increased to improve association of two or more moleculescompared to ambient conditions. It is possible to combine the variousaspects of the process described herein by first subjecting a mixture toheat and mixing in order to separate a ligand or analyte in the mixturefrom endogenous binding partners in the mixture. The temperature,mixing, or both, are changed from the initial condition to enhanceligand complex formation with an exogenously supplied binding partnerrelative to ligand/endogenous binding partner complex formation atambient temperature and mixing. Generally, the second temperature and/ormixing conditions are intermediate between ambient conditions and theconditions used in the first separating step above. At the secondtemperature and mixing condition, the separated ligand is reacted withthe exogenously supplied binding partner.

Polymerase Chain Reaction (“PCR”) Thermal Cycling

One of the bottlenecks of the PCR technique is cooling time. The heatingcycle is rapid; however, cooling is limited by convection. Even inbiochip formats, in which DNA or another target molecule is immobilizedin an array on a microdevice, there is no “active” cooling process.However, certain embodiments of the invention can be used to overcomethis bottleneck.

In certain embodiments, a treatment process can be used to both heat andcool the sample rapidly with little overshoot from a baselinetemperature at which the primer and target to be amplified anneal. Theprocess can be summarized as follows. A sample is treated withrelatively high power sonic energy such that the sample absorbs sonicenergy and is heated. Then, the sample is mixed at low power to cool thesample by forcing convection, which may be accomplished in conjunctionwith a cool water bath. In some embodiments of the apparatus, the systemis a “dry top” system, that is, a system in which a microplate,typically with its top temporarily sealed with plastic film, floats onor is partially immersed in a controlled-temperature bath. In thisarrangement, the PCR reaction may be monitored in real-time fortemperature using, for example, an infra-red detection probe, and forreaction products by examining the incorporation of fluorescent dyetagged nucleic acid probes into the PCR product. This “dry top” systempermits real-time analysis and control of the process. Information fromthe temperature sensor can be used in a feedback loop to control theduty cycle of the acoustic input, such as the number of bursts/second,or otherwise control the amount of heating. Also, fluorescence from anintercalated probe can provide a computer with information on whichwells have reached a certain point in the reaction, such as when aparticular level of fluorescence is sensed, allowing, for example, thecomputer to control application of sonic energy or sample location suchthat certain wells are skipped in the processing cycle until other wellshave attained the same point in the reaction or that certain wells arenot processed further.

G. Purification, Separation, and Reaction Control

Focused sonic fields can be used to enhance separations. As notedelsewhere, sonic foci can be used to diminish or eliminate wall effectsin fluid flow, which is an important element of many separationprocesses, such as chromatography including gas chromatography, sizeexclusion chromatography, ion exchange chromatography, and other knownforms, including filed-flow fractionation. The ability to remotelymodulate and/or reduce or eliminate the velocity and concentrationgradients of a flowing stream is applicable in a wide variety ofsituations.

Sonic fields also can be used to minimize concentration polarization inmembrane processes, including particle classification, filtration offine particles and colloids, ultrafiltration, reverse osmosis, andsimilar processes. Concentration polarization is the result of thetendency of filtered material to be present at high concentration in alayer on the filter. This layer has a low fluid concentration and, thus,diminishes the rate of filtration as the filtered solution becomes moreconcentrated, or as the layer thickens. This layer can be stirredremotely by focused sonic energy of low to moderate intensity. Flowrate, thus, can be enhanced without significant cost in energy ormembrane life.

Such sonic energy fields can be used to enhance reaction rates in aviscous medium, by providing remote stirring on a micro scale withminimal heating and/or sample damage. For example, some assays rely onthe absorption of analytes by reagents, such as antibodies, which arebound to macroscopic particles. In a viscous fluid to be analyzed, suchas sputum or homogenized stool, the ability to stir such a sampleremotely, aseptically, and essentially isothermally can significantlydecrease the time required to obtain equilibrium of the analyte with thereagents on the particle.

Likewise, any bimolecular (second-order) reaction where the reactantsare not mixed at a molecular scale, each homogenously dissolved in thesame phase, potentially can be accelerated by sonic stirring. At scaleslarger than a few nanometers, convection or stirring can potentiallyminimize local concentration gradients and thereby increase the rate ofreaction. This effect can be important when both reactants aremacromolecules, such as an antibody and a large target for the antibody,such as a cell, since their diffusion rates are relatively slow anddesorption rates may not be significant.

These advantages may be realized inexpensively on multiple samples in anarray, such as a microtiter plate. The use of remote sonic mixingprovides a substantially instantaneous start time to a reaction when thesample and analytical reagents are of different densities, because insmall vessels, such as the wells of a 96 or 384 well plate, littlemixing will occur when a normal-density sample (about 1 g/cc) is layeredover a higher-density reagent mixture. Remote sonic mixing can start thereaction at a defined time and control its rate, when required. Thestepping and dithering functions allow multiple readings of the progressof the reaction to be made. The mode of detecting reaction conditionscan be varied between samples if necessary. In fact, observations bymultiple monitoring techniques, such as the use of differing opticaltechniques, can be used on the same sample at each pass through one ormore detection regions.

H. Further Uses for Remotely Actuated and Controlled Solution Mixingwith Sonic Energy

Control of sonic energy emission, sonic energy characteristics, and/orlocation of a target relative to sonic energy also can be used to pumpand control the flow rate of liquids, especially in capillaries; enhancechemical reactions, such as enhancing second-order reaction rates;increase effective Reynolds number in fluid flow; and control thedispensing of semi-solid substances.

By focusing sonic energy and positioning it near a wall of a vessel, awall of a tube, or another discontinuity in a fluid path, many localdifferences in the distribution of materials within a sample and/orspatially-derived reaction barriers, particularly in reactive andflowing systems, can be reduced to the minimum delays required formicroscopic diffusion. Put differently, enhanced mixing can be obtainedin situations where imperfect mixing is common. The range of thesesituations is illustrated below.

Control of Flow Rates of Fluids

Miniaturization of analytical methods, such as analysis on a chip,require concomitantly miniature capillary-sized dimensions for fluidflow paths. Sonic excitation provides a convenient, simple, and sterilemanner to accelerate flow in capillaries. During excitation, the fluidis locally turbulent, and so flows more readily. By selective or timedlocal sonic excitation, optionally controlled with a feedback loop, therate of flow through complex microfluidic paths can be remotelymanipulated in a controlled manner.

Increase of Effective Reynolds Number in Fluid Flow

At low Reynolds numbers, the velocity profile of laminar fluid flow in apipe or other conduit is approximately parabolic. Fluid at the center ofthe pipe is flowing significantly faster than fluid near the wail.Therefore, conversion of fluid carried in the pipe from one fluid toanother is quite slow, and, in principle, infinitely slow.

This effect effectively vanishes at higher Reynolds numbers becauseturbulence mixes the fluid at the center with fluid at the peripheryvery rapidly, so that composition differences are rapidly eliminated.However, there are significant disadvantages to operating a fluidconduit under turbulent conditions, including high backpressure andcorrespondingly high energy expenditure.

If sonic energy is focussed in, on, or near the wall of the pipe, nearthe fluid/wall boundary, then local turbulence can be obtained without ahigh rate of bulk fluid flow. Excitation of the near-wall fluid in acontinuous, scanned, or burst mode can lead to rapid homogenization ofthe fluid composition just downstream of the excited zone. This willsharpen the front between any two fluids passing through a pipe insuccession.

This effect is useful in several areas, including chromatography; fluidflow in analytical devices, such as clinical chemistry analyzers; andconversion of the fluid in a pipeline from one grade or type to another.Since most of the effect occurs in a narrow zone, only a narrow zone ofthe conduit typically needs to be sonically excited. For example, insome applications, the focal zone of the sonic energy can be the regionclosest to a valve or other device which initiates the switch ofcomposition. In any of these applications, feedback control can be basedon local temperature rise in the fluid at a point near to or downstreamof the excitation region.

Enhancement of Second-Order Reaction Rates

Microsonication can be used to speed up, or to homogenize, the rate ofchemical reactions in a viscous medium. The flow of individualmolecules, and of heat, is generally slower in a more viscous medium.For example, it is more difficult to mix molasses with water than to mixvinegar with water. Similarly, in an aqueous solution, it becomesincreasingly difficult to maintain the rate at which soluble monomersundergo a polymerization reaction, forming a soluble polymer, as themolecular weight of the polymer increases with each added monomer,because the viscosity of the solution increases.

Mixing of molasses and water with a stirrer is simple, but not easilysterile, and a polymer can be degraded by shear caused by stirring witha stirrer. Focussed sonication can readily mix pre-sterilized liquids ina remote manner without contamination. Focused sonic energy also can mixpolymerizing materials without application of macroscopic shear, and socan minimize shear degradation of the formed polymer. Similarly, apolymerase chain reaction can be accelerated by brief pulses of sonicenergy, or by longer pulses which also provide the desired temperatureincreases, to prevent the retardation of the reaction due to localdepletion of the nucleotide triphosphate monomers.

Controlled Dispensing of Semi-Solid Substances

Highly viscous liquids, including materials which effectively act assolids or near-solids, can flow at an increased rate when sonicallyexcited by a remote or local sonic source. This excitation may be underfeedback control. This effect can be caused by local reduction ofimpedance to flow by walls of a conduit, as described above, and bylocal heating from sonic energy input. As a simple example, theeffective viscosity of an ink jet ink, and thus the rate of itsdelivery, can be controlled by focused, localized sonic energy delivery.Analogous uses are possible wherever the viscosity of a fluid, includinga semi-solid or a solid capable of melting, is significant. Likewise,flow of particulate materials in a fluid where the particles areinsoluble in the fluid can be selectively stimulated to occur, or beaccelerated, with focussed, controlled sonic waveforms.

Various embodiments of the present invention will be further understoodby reference to the following non-limiting examples.

IV. EXAMPLES Example 1 Isolation of Intracellular Components from Cells

To further aid in the understanding of this invention, a procedure forthe isolation of intracellular components from cells imbedded in amatrix is described. A sample of about 100 cu. mm. volume is placed intoeach well of a multiwell plate, such as a 96 well plate, having acapacity of about 200 microliters (200 cu. mm). The entire plate is thenfrozen and reduced to about minus 40° C. Then, about 100 microliters ofan extraction solution, precooled to 4° C., is added to each well whilethe plate is held at minus 40° C. This maintains the sample temperatureat minus 20° C. or less, while providing a smooth surface in the wellfor coupling to the wave source. A sheet of flexible plastic foil isoptionally affixed to the plate to prevent cross contamination betweenthe contents of the wells, or between the wells and the wave source. Apiezoelectric wave source is provided and positioned on the plate. Thesource has 96 pins in the appropriate array, and each pin is connected,preferably removably, to a piezoelectric driver carried in a commonholder for the pins; the drivers,.and associated circuitry. Then, aseries of electrical pulses is applied to the drivers to generate shockwaves in the samples. The application of the series is preferably drivenby an automated controller, such as a custom chip or a programmedcomputer. The wave source is removed, preferably robotically, and theplate is rapidly warmed to 4° C. The 96 solutions in the wells of theplate are agitated by a mild sonic vibration, at an intensity too low tomechanically damage the target molecules. After a defined incubationperiod, such as 30 seconds, the plate, still bearing the plastic foil,is removed to a centrifuge to pellet debris, and the top 50 microlitersof each sample is removed for further analysis.

Example 2 Steady-State Temperature Control with Controlled WaveformGeneration to Uniformly Mix a Sample

In this example, the duty cycle of an ultrasound treatment was varied toreduce steady-state temperature rise within a sample, compared to acontinuous wave, 100% duty cycle.

A. 1.1 MHz high power transducer was applied to a sample treatmentvessel. The sample treatment vessel was constructed of two layers ofthin film. The bottom consisted of a ⅜ inch (9.5 mm) diameterhemispherical “bubble” from bubble-wrap packing material with the flatside cut away to yield a ⅜ inch diameter hemisphere, made of a thinplastic. The top layer was 0.001 inch thick saran film again, a thinplastic. The vessel, having approximately a 300 μl total volume,contained a liquid sample of 50% methanolic solution, that is 1:1methanol volume to water volume. The sample treatment vessel wasinserted into a frame that allowed the bubble to protrude into the focalzone of the transducer in a water bath. The vessel was then placed intoa water bath at 3.5° C. The saran film top side was exposed to the air.The temperature of the internal liquid was measured with a J-typethermocouple at the periphery of the treatment vessel and a thermocouplemeter, Omega model #DP116-JC2. An input signal of 500 mV sine waves at1.1 MHz frequency generated by an arbitrary waveform generator input toa 55 dB RF amplifier was applied to the transducer resulting in a peakpositive pressure of approximately 15 MPa and a peak negative pressureof approximately −6 MPa in the focal zone of the resulting acousticfield. The transducer was focused on the sample vessel containing themethanolic solution such that the sonic energy entered the vesselthrough the bottom film made from the bubble wrap and converged withinthe vessel. The acoustic dosage received by the sample was 1,000cycles/burst, 10,000 bursts per dose for a total dose of 10,000,000cycles. The same sample was treated with duty cycles of 1%, 5%, 10%, and20%.

A steady state condition was obtained after an initial transienttemperature change. In all cases the transient temperature changeoccurred within the first 30 seconds and the temperature became stablefor the remainder of the dose, up to several minutes, depending on theduty cycle. The data are presented in Table 1, below.

TABLE 1 Temperature rise in degrees Celsius as a function of duty cycleand amplitude. Duty cycle Temperature Rise at 500 mV Temperature Rise at750 mV  1% 0.6 1.1  5% 1.1 2.7 10% 2.0 4.9 20% 2.7 6.4

This example demonstrates that high intensity focused ultrasonic energycan be focused on an in vitro sample without deleterious heatgeneration. The sample treatment vessel described here is optimized forefficient heat transfer and acoustic transparency. By providing areliable way to monitor the status of the sample, such as an infraredsensing temperature probe, and control over the electrical waveforminput to the ultrasound transducer, the ultrasound signal can beoptimized to maximize energy transfer while minimizing temperature riseor other deleterious effects.

Example 3 Increasing Extraction Output by Using Infrared TemperatureFeedback to Vary Either the Duty Cycle and/or the Voltage

A 1.1 MHz high power transducer was configured to treat a sample in atreatment vessel constructed in a manner as described in Example 2. Theacoustic dosage received by a sample of leaf tissue suspended in amethanol solution in the vessel was 500 cycles/burst, 2,000 bursts perdose, with a variable duty cycle. The starting temperature of the vesselwas less than 1° C.

Upon initiation of the treatment, the temperature within the vesselstabilized within 10 seconds and remained stable during the dosageinterval of up to ten minutes. The duty cycle was adjusted to controlthe temperature rise. The effect of the dose was visually similar,whether the dose was received by the sample as one long burst in acontinuous wave (“CW”) or as an accumulation of shorter bursts, having aduty cycle less than 100%.

The results are shown graphically in FIG. 8. At a 500 mV wave amplitude,the temperature of the treated sample rose approximately 0.5° C., 1.9°C., 2.8° C., and 3.0° C. from a starting temperature of approximately0.0° C. at duty cycles of 1%, 5%, 10%, and 20%, respectively. At a 750mV wave amplitude, the temperature of the treated sample roseapproximately 1.1° C., 2.7° C., 4.9° C., and 6.4° C. from a startingtemperature of approximately 0.0° C. at duty cycles of 1%, 5%, 10%, and20%, respectively. These data are useful for constructing a sonic energycontrol system either with or without a feedback loop.

Example 4 Sample Mixing and Disruption with Synchronized Intra-SampleFocal Zone Positioning

This example indicates that movement of a sample through the sonicenergy field has beneficial effects. When a focused ultrasonic dose withnon-optimized mixing waveforms was applied to a leaf tissue sample thatcontained a heterogeneous mixture of leaf lamina, stalks, veins, andpotting soil, a small portion of the sample was disrupted. With anultrasonic dose and a stationary focal zone, the larger particles ofleaf clumps, debris, etc. were visibly “pushed” to the peripheral edges,while the smaller particles remained within or near the focal zone. Whenthe focal zone was swept across the sample slowly in a circular motionduring the treatment, clumps of material around the periphery of thetreatment vessel were brought into the focal zone and were visiblybroken up. Manually moving the sample across the focal zone with aNewport Series 462 x, y positioning stage resulted in better resultsthan with a stationary focal zone. However, while manually moving thesample was beneficial for treatment purposes, the manual moving resultswere not as precisely repeatable as they can be with acomputer-controlled positioning system such as was used in Example 5,below.

A benefit of automated movement of the sample relative to thetransducer, also known as x-y dithering, can be to prevent a bubbleshield from forming and blocking cavitation within the sample treatmentvessel. Another benefit is that the x-y dithering can also enhancetreatment of sample suspensions that have a high viscosity and do notmix well. Dithering becomes increasingly advantageous as the sampletreatment vessel becomes significantly larger than the focal zone.

The automated movement of the sample relative to the ultrasoundtreatment can be advantageous with an unfocused transducer, such as a 20kHz cell disrupter ultrasonic probe, because, for example, relativemotion between the sample and the ultrasound source during treatmentprevents the suspended particles in the sample from collectingpreferentially at the low intensity nodes of the acoustic field.

Example 5 Extraction of Amino Acids from Plant Leaf Tissue withUltrasound and Positional Dithering

Samples of approximately 100 mg of A. thaliana leaf tissue werecollected and flash frozen in liquid nitrogen. The frozen material wasstored in 2 ml vials at −75° C. until the day of the experiment. Thesamples were then transferred to dry ice and placed into individualvessels for treatment. The treatment vessels consisted of ⅜ inch (9.5mm) diameter hemispherical “bubbles” made from bubble-wrap packagingmaterial, as described in Example 2. The tissue samples were placed intothe sample treatment vessels with approximately 200 microliters ofpre-chilled, 4° C. 90% methanol:water (9:1, v/v). The samples were thenwarmed to approximately 4° C. for the treatment.

The apparatus contained a transducer in a water bath at roomtemperature, approximately 24° C., with the treatment vessel ultimatelypositioned in another inner water bath having an acousticallytransparent film located in the path of the converging acoustic waves.The inner water bath was chilled with copper coils to about 4-6° C. Thesample treatment vessels were inserted into a computer-controlled x, y,z positioning system. The samples were then aligned in the focal zone ofthe transducer, using predetermined positions, with the beginning of thefocal zone convergence at approximately 2 mm inside of the treatmentvessel.

Four conditions were tested during the experiment. First, anexperimental condition (type one) was tested, where a leaf tissue samplewas placed in a methanol solution, subjected to sonic energy,centrifuged, and the supernatant removed. This supernatant was testedfor the presence of amino acids, peptides, proteins, and other primaryamines (“amino acids”). The pellet was resuspended in methanol,vortexed, centrifuged, and the supernatant removed. This secondsupernatant was tested for the presence of amino acids. The amount ofamino acids recovered from the first “extraction” step was divided bythe total amino acids extracted during both extractions to determine thefraction of amino acids recovered during the first extraction of thetotal number of amino acids present. Second, an experimental condition(type two) was tested, similar to the first experimental condition,except that during sonic energy exposure, the sample was dithered, beingmoved relative to the sonic energy source.

The waveform used in all of the treatments in this example had anamplitude of 500 mV, a frequency of 1.1 MHz, bursts of 1000 cycles and aduty cycle of 10%. This waveform was previously found to effectivelytreat and mix the sample without generating excessive heat, as describedabove. All of the samples received 10,000 of these bursts.

The sample was continually mixed during the treatment. The pre-definedtreatment parameters insured that both the disruption phase and mixingphase occurred at constant temperature. From other experiments, it isknown that the temperature remained essentially constant and wasessentially isothermal with the inner bath. The inner bath temperatureranged from 4-6° C., and the sample temperature also ranged from 4-6° C.during the process.

Immediately following the experimental treatment, as much of the sampleas possible was transferred to 0.5 ml polypropylene vials. The typicalrecovery was greater than 75% of the liquid and solid sampletransferred. The samples were then gently vortexed for 10 seconds, andwere centrifuged for 2 minutes at 5,000 rpm with a smallmicrocentrifuge. The supernatant was immediately transferred to anothermicrocentrifuge tube and was placed in a −80° C. freezer until analysis.

There were two controls. First, a sham control was used that wasidentical to the first experimental condition, but without treatmentwith ultrasonic energy. The RF amplifier was not turned on. Second, amethanol double extraction control process, such as one that might beused for extraction without treating a sample with sonic energy, wasutilized to compare with the ultrasonic treatment process. The controlprocess was to add a 90% methanol solution to the plant tissue, let themixture stand at room temperature for 2 hours, vortex the mixture forone minute, centrifuge the mixture for 5 minutes, remove the supernatantin a first extraction and perform an amino acid assay. Methanolextraction was repeated on the pellet that remained after thesupernatant was removed during the first extraction step. Again, thesupernatants from the first and second extractions in all of thecontrols were tested for amino acid content and the amino acid contentwas expressed as a fraction of the total amount of amino acids collectedduring the first and second extractions, combined. All control samplesunderwent one freeze/thaw cycle, just as the two experimental conditionsdid, and, consequently, there was tissue lysis and amino acid releasedue to the mechanical freeze/thaw effects. Thus, this freeze/thaw effectis controlled for in the results.

Typically, in either of the two experimental conditions, about 10 μmoleof amino acids per wet gram of tissue was recovered from the firstextraction and about an additional 3-5 μmole of amino acids per wet gramweight of tissue was recovered from the second extraction. Allextractions were performed in duplicate or triplicate.

The extract samples, namely the supernatant removed during bothextractions during both of the experimental conditions and both of thecontrol conditions were assayed for total amino acids with afluorescamine assay. The fluorescamine reacts to form stable fluorescentsubstances with amino acids, peptides, proteins, and other primaryamines under mild reaction conditions. Fluorescamine (0.07 ml, 14.5 mM)was added to 0.08 ml of triethylamine/acetate (pH 8.6), and this mixturewas added to 20-50 μl aliquots of the extract sample and, subsequently,was brought up to a total volume of 200 μl with 100% methanol in amicrotiter plate. The mixture was incubated for 30 minutes at 25° C. Themicrotiter plate was then excited at 395 nm and the emission detected at460 nm in a standard fluorescent plate reader. Standard curves includeda mixture of phenylalanine, alanine, and leucine in 10% acetonitrile inthe range of 0.1 to 10 mM, such that the fluorescent signature of theextract samples could be compared with a standard to determine the molarconcentration of amino acids in the samples.

The results are expressed as micromole of amino acids per gram of wettissue for both first and second extractions. The effectiveness of thetreatment is expressed as the first extraction as a percentage of thetotal extracted amino acids in both extractions.

The experimental results, are shown in Table 2, below, and these resultswere averaged for comparison, as shown in Table 3. The results indicatethat: (1) the control process samples of fresh frozen tissue extractedwith a 90% methanolic solution that were not treated with focused sonicpulses, but were vortexed, centrifuged and assayed as for theexperimental samples, resulted in approximately 82% of the total aminoacids being present in the first extraction; (2) the sham-controlsamples which were inserted into the treatment vessel, incubated at 4°C. for eight minutes (equal to the longest treatment interval),vortexed, and centrifuged resulted in approximately 85% of the totalamino acids being in the first extraction; (3) experimental samples(type one) that were exposed to an ultrasonic dose of 500 mV, 1,000cycles/burst, 10,000 bursts, and a cumulative dose of 10 million cycles,without dithering, resulted in approximately 94% recovery of total aminoacids in the first extraction with a coefficient of variation of lessthan 2%; and (4) experimental samples (type two) that were treated witha similar dosage as with type one, with the addition of positionaldithering of the sample relative to the ultrasound source during thetreatment process, resulted in greater than 99% recovery of total aminoacids in the first extraction, with a less than 1% coefficient ofvariation. The experimental samples (type one and two) appeared greenand cloudy like “pea-soup” following treatment, whereas the shamcontrols were clear and tinged green with chlorophyll.

In the experimentally processed samples, stems did not appear to beaffected by the process; however, leafy tissue was visibly disrupted asa result of the treatment. The process was rapid, reproducible, andrequired less hands-on time than the control process. Typically, toobtain the same amount of material from the tissue as with theexperimental process, without the use of sonic processing, would requirethree or more repeated methanol extractions using the control process.As the sample is often at room temperature during this process, labilecellular constituents may be degraded.

TABLE 2 Sample data for various treatments. Amino acids extracted in thefirst extraction as a Sample Number percent of the total amount(randomly assigned) Treatment Description of extracted amino acids 4Control process 86.5 10 Control process 75.9 34 Control process 82.7 11Sham control 83.1 22 Sham control 92.4 31 Sham control 78.2 9 Mix, typeone 90.7 49 Mix, type one 96.8 16 Mix/dither, type two 99.2 46Mix/dither, type two 99.9

TABLE 3 Averaged results of Table 2. Average amino acids extracted inthe first extraction as a percent of the total amount of extracted aminoacids (an average of the results for each treatment as shown CoefficientTreatment in Table 2) of variation Control process (3 samples) 81.7%6.6% Sham control (3 samples) 84.6% 8.5% Mix, type one (2 samples) 93.8%4.6% Mix/dither, type two (2 samples) 99.6% 0.5%

Example 6 Controlled Sample Mixing with Control of Both Cooling andHeating

This example illustrates ultrasound-forced convective cooling of liquidsamples. The experimental apparatus used in this example was similar tothat used in Example 5, above. The waveform input to the transducerconsisted of 10,000 1.1 MHz frequency bursts with 1000 cycles per burstand a 10% duty cycle. The amplitude was 500 mV (into a 55 dB RFamplifier). The cumulative dose was 10 million cycles. This waveform wasgenerated by LabVIEW software driving two function generators operatingin series. This is the “mix-and-treat” waveform used in Example 5,above. The sample vessel was a polypropylene vial with the ends replacedwith polyethylene film and held in the focal zone by a fixture. Thesolvents used were either water or a 90% methanol solution. The samplevessel was filled to minimize headspace. The temperature was monitoredwith a ColePalmer, Model 39670-00 infrared sensor with a spot size of0.17″ (4.3 mm) at 0.0 inches distance and 1.0″ (2.5 cm) at 5 inches(12.7 cm) distance. The sensor was connected to an Omega Model DP116-JC2temperature display. The sensor, with a response time of less than 450milliseconds, was in close proximity with the sample top.

Treatment vessels were placed into a water bath at 3.5° C. Beforetreatment, the samples were allowed to equilibrate at a temperatureintermediate to the bath temperature and the ambient air temperature.Upon initiation of ultrasound treatment, the temperature of the samplesdropped from their initial temperature to a temperature near that of thewater bath and then rose above the initial temperature. This observationindicates that for the first few seconds of the treatment, a net coolingeffect was achieved by forcing convective heat transfer with ultrasonicenergy. The data are shown in Table 4, below. In the case of themethanol solution, the temperature was depressed below the startingequilibrium position for the first 40 seconds. In the case of the water,the temperature was depressed for the first 10 seconds.

TABLE 4 Sample temperature during treatment (degrees Celsius) as afunction of the fluid in the sample treatment vessel and treatment time.Bath temperature, in which the treatment vessels sit, is 3.5 degreesCelsius. Temperature of Temperature of sample in degrees sample indegrees Treatment Time (seconds) Celsius (90% methanol) Celsius (water)0 6.5 6.5 5 4.6 4.5 10 3.8 5.3 15 3.9 6.8 20 4.4 7.8 25 5.0 8.2 30 5.68.4 35 5.9 8.5 40 6.4 9.1 45 7.2 9.8 50 7.4 9.8 55 7.8 10.0 60 8.5 10.265 9.4 10.5

These samples were treated with a powerful treatment waveform that wasdeveloped to disrupt tissue samples. The waveform likely can be modifiedto preferentially enhance the cooling effect such that the sampletemperature would be depressed below the equilibrium temperature for aslong as necessary. Other waveforms can be optimized for heating and fortreating the sample. In this way, the sample could be sequentiallyheated, cooled, and/or treated. Both the heating and cooling waveformspromote mixing. This allows acceleration and control of reactions thatwould otherwise be rate-limited by diffusion. For example, an enzymaticreaction occurring slowly in a cold solution, such as one at 4° C., maybe activated by application of a heating waveform. Following cessationof the heating waveform, the sample is rapidly chilled by a coolingwaveform to inhibit the reaction. This rapid temperature cycling isuseful for thermal-cycle based protocols such as polymerase chainreaction (PCR).

Example 7 Passive Cavitation Detection (PCD) to Monitor Efficiency ofUltrasonic Dosage

An apparatus can be assembled to measure cavitation induced by anultrasonic wave. One possible configuration uses a Panametrics A315R-SU,10 MHz transducer, optionally a Kron-hite 23 band-pass filter, aPanametrics 5676, 20 MHz, 40 dB pre-amplifier, a Panametrics 5607 gatedpeak detector, and a National Instruments, PCI-6111E, 5 MHz, two channelanalog acquisition card digitizer board. LabVIEW instrument controlsoftware is configured to analyze the signal produced by the gated peakdetector. This is considered “passive” cavitation detection, because itdetects acoustic signals generated directly by the motion and collapseof cavitation bubbles. Other useful active cavitation detection systemsare based on the scattering or modulation of laser light by cavitationbubbles.

Cavitation bubble collapse generates wide-band noise. Bubbles are veryeffective scatterers of ultrasound. The pulsation mode of a bubble isreferred to as monopole source which is a very effective acousticsource. For small, generally linear oscillations the bubble simplyscatters the incident acoustic pulse. However, as the response becomesmore nonlinear, it starts also to emit signals at higher harmonics. Whendriven harder the bubbles start to generate subharmonics as well.Eventually, as the response becomes aperiodic or chaotic, the scatteredfield tends towards white noise. In the scenario where inertialcollapses occur, a short acoustic pressure pulse is emitted. An acoustictransducer can be configured to detect these emissions. There is astrong correlation between the onset of the emissions and cell lysis.

The PCD is normally arranged to be confocal with a high power transducerso that it collects cavitation signals from the focus of the high powerbeam. The signal from the PCD is amplified and passed through a 2 MHzhigh-pass filter. The high-pass filter removes the 1 MHz signal due toscattering of the fundamental pulse and any other scatterers. The amountof cavitation that the sample has been subjected to can be estimated byintegrating the noise signal received by the PCD.

The signal generated by the cavitation detection system can be used as afeedback control element in an automated system. The automated systemcontrols the cavitation by either manipulating the sample position bydithering or other motion to affect the position of cavitationnucleation sites, modulating or controlling the ultrasound signal,modulating or controlling overpressure (as in Example 8), and/orcontrolling the composition of dissolved gasses in the treatment vessel.

Example 8 The Application of Overpressure to Limit or Control Cavitation

A treatment vessel was overfilled with fluid prior to sealing thevessel. The interior fluid chamber was at a slight overpressure relativeto atmospheric pressure and the water bath pressure. This overpressureinhibited cavitation effects, such as tissue disruption within thesample vessel. When a sample of leaf tissue was placed in this settingand treated with the previously described experimental apparatus with awaveform consisting of 500 mV amplitude, 500 cycles/burst, 1,000 bursts,and 10% duty cycle input to a 55 dB amplifier, there was only a slighttissue disruptive effect. When the voltage was increased to 700 mV withthe same dosage parameters, there was no marked change in tissuedisruption.

When the sample vessel was opened to relieve the overpressure and thesample was given a 500 mV dose, the tissue was disrupted. Theoverpressure apparently inhibited the cavitation bubble formation andcollapse that is related to and can cause tissue disruption. This resultdemonstrates that overpressure may be used to control or limitcavitation. Overpressure can be effectively integrated with apredetermined pattern of sonic energy exposure, both by altering thesonic energy and the location of the sonic energy relative to the sonicenergy focal zone, such that the disruptive effects of sonic energyexposure due to cavitation can be selectively muted with pressurecontrol. Additionally, in conjunction with a cavitation sensor thatprovides information to a control system through a feedback controlloop, controlled overpressure could be used to treat biological or othermaterials where it is desirable to control the intensity of thecavitation, such as in the controlled disruption or permeabilization ofbiological membranes.

Example 9 Treatment Vessel Design-Shape, Wall Thickness, and MaterialChoice

Several factors can be relevant in the design of a treatment vessel. Thetreatment vessel geometric design shown in FIG. 5A depicts a device witha dome shape that is able to transfer the heat generated from theultrasound process from the sample mixture to the surrounding waterbath. Example 2, above illustrates the use of this dome-shaped deviceand the importance of good heat transfer and acoustic transparencycharacteristics. Typically, the material from which a treatment vesselis constructed should absorb relatively little sonic acoustic energy andshould impede sonic energy at a level that is similar to the sonicenergy impedance of water. The thickness of the material also should berelatively thin, for maximizing ultrasound transmission, maximizing heattransfer between the interior and exterior of the vessel, andfacilitating monitoring of the treatment vessel and it contents, by, forexample, infra-red temperature sensors, cavitation detection sensors,and/or video or optical monitors.

Standard polystyrene or polypropylene microwell plates, such as 96 wellplates, have wall and bottom thickness of approximately 1 mm. Tests witha microwell plate, oriented in a horizontal plane, that is exposed tosonic energy from a needle-tip transducer hydrophone with the acousticpath completely submerged, resulted in approximately 70% transmissionthrough polystyrene. The resultant absorption is significant in a highpower dosage. For example, a 1:1 MHz continuous wave sample dosage of500 mV input to a 55 dB RF amplifier for 30 seconds applied to apolypropylene microwell plate generates enough heat to bring 300microliters of ice to a boil within seconds.

Temperature rise was measured in two different vessels, the“bubble-wrap” vessel described in Example 2 and a polypropylene vial,using a mix-treat waveform of 10% duty cycle, 10,000 bursts, 1,000cycles/burst at 500 mV as described in the examples above. Variousvolume/volume ratios of methanol in water were evaluated, including 0%methanol, 50% methanol, 90% methanol, and 100% methanol. In each case,the starting temperature was close to 1° C. The results shown in Table 5are the magnitudes of temperature rise following dosage. The temperaturewas measured with the infrared sensor and meter, as described in theexamples above.

TABLE 5 Temperature rise in degrees Celsius as a function of methanolconcentration and sample vessel type. Temperature Temperature rise ofmethanol rise of methanol solution in “bubble- solution in Methanolsolution (volume wrap vessel” polypropylene vial methanol/volume water)(degrees Celsius) (degrees Celsius)  0% 0.0 4.4  50% 2.2 6.3  90% 5.47.2 100% Not determined 10.5

These results show that the bubble-wrap vessel of Example 2 is bettersuited to samples that should remain substantially isothermal duringtreatment than is a polypropylene vial.

Example 10 Sonic Energy Optimization

A series of experiments was performed using the apparatus describedabove to optimize exposure of a sample to sonic energy. First, awavetrain was optimized for both treatment and mixing. Briefly, and asmore fully described in Example 2, a sample treatment vessel wasconstructed from a ⅜ inch (9.5 mm) diameter hemispherical “bubble” takenfrom bubble-wrap packaging material. The flat side of the bubble was cutopen with a hot-knife to access the interior. The sample treatmentvessel was held in a metal fixture such that the focal zone of theacoustic field was within the vessel. The sample treatment vessel had avolume of approximately 300 microliters.

A. thaliana leaf tissue was prepared by freezing, lyophilizing, andgrinding. Approximately 25 milligrams (dry weight) of tissue was putinto the sample treatment vessel. This tissue was rehydrated with 200microliters of a 50% MeOH solution. A layer of plastic saran film 0.001inches (0.025 mm) thick was positioned on the flat side of the treatmentvessel and the whole was clamped in a metal fixture.

Several different acoustic wavetrains were applied to the sample. Themixing effect was judged visually. The temperature rise was measuredwith a J-type thermocouple at the periphery of the treatment vesselusing an Omega DP116-JC2 meter. The parameters being varied include thenumber of cycles per burst and the duty cycle. Fixed parameters includedthe 1.1 MHz frequency of the cycles and the 500 mV amplitude applied toa 55 dB RF amplifier and thereafter to the transducer. The results ofvarying the parameters are shown in Table 6, below.

TABLE 6 Results of varying the number of cycles per burst and the dutycycle on heating of the sample and mixing of the sample. Cycles perTemperature Rise of Sample Mixing Effect Burst Duty Cycle in degreesCelsius in Sample 5  1% 1.5 none 500 10% 3.5 none 10,000 20% 6.0 slightstirring 10,000 10% 4.0 some stirring 1,000 10% 3.2 good mixing

Repeated trials of the final combination, 1,000 cycles per burst and 10%duty cycle, showed that this combination is effective at mixing thesample material in the sample treatment vessel. This combination ofparameters produces a compromise wavetrain that both mixes and treatsthe sample.

Further experimentation verified the ability to separate the heating andmixing functions, such that each function can be optimized independentlyof the other and apparatus and methods of the present invention canalternate between these two functions. More particularly, a treatmentwavetrain was alternated with a mixing wavetrain. In this series ofexperiments, the sample was approximately 200 micrograms of fresh-frozenA. Thaliana in 600 microliters of water. The sample treatment vessel wasa polypropylene cylinder with an inside diameter of 0.5 inches (1.3 cm)and a length of 1.7 inches (4.3 cm). The open end of this cylinder wascovered with 0.001 inch (0.025 mm) thick polyethylene film as anacoustic window. The treatment vessel was positioned such that the focalzone of the sonic energy was inside the treatment vessel and themajority of the acoustic energy passed through the polyethylene filmwindow.

The treatment wavetrain was 5 cycles per burst, a 1% duty cycle, 500,000total cycles, and a 500 mV amplitude into the RF amplifier andthereafter to the transducer. The mixing wavetrain consisted of CWultrasound at 100 mV for 500,000 cycles. The treatment wavetrain wasfollowed by the mixing wavetrain. Each wavetrain takes about 0.5 secondsto complete. The treatment wavetrain followed by the mixing wavetrainwas repeated 5 times. Mixing was determined by examining the sampleduring treatment to visualize tissue particles moving in the treatmentvessel. Treatment was determined by examining the tissue samples under astereo microscope for the creation of small tissue particles and/or theshredding of larger tissue particles.

The mixing wavetrain was effective and good mixing of the sample wasvisually apparent. The treatment wavetrain was partially effective; thesmaller fragments of tissue were treated but the larger ones were notsubstantially treated. This experiment demonstrates that a treatmentwavetrain can be alternated with a mixing wavetrain to achieve bothtreatment and mixing. Alternating treatment and mixing wavetrains allowseach to be optimized for its specific function.

Example 11 Sonolysis of Plant Leaf Tissue

A 70 mm diameter focused ceramic piezoelectric transducer dome wasinserted into a water tank and the focal point domain was defined to beapproximately 62 cm away from the surface of the dome. A continuous,sinusoidal wave form of 1 MHz frequency with 0.2 Volt from thepreamplifier with a resultant 5 MPa positive pressure from the focusedpiezoelectric transducer was generated for short time durations of 1 and10 seconds. The focused energy zone was approximately 3 mm diameter by 6mm in length. Approximately 100 mg of Arabidopsis thaliana leaf tissuehad been collected and immediately frozen in liquid nitrogen and storedat −70° C. until use. Tissue samples were inserted into 0.325 ml of CTABbuffer (1M TRIS pH 7.5, 200 ml, CTAB (Hexadecyltrimethyl AmmoniumBromide) 20 g, NaCl 81.76 g, 0.5 M EDTA pH 7.5 40 ml, H₂O 1,500 ml) intoa standard flat-bottom, polystyrene 96-well microtiter plate (Immulon1B, cat. 3355, Dynex Technologies, Chantilly Va.). In some otherprotocols, 10 μl 2-mercaptoethanol/ml CTAB buffer is added prior to use.For this experiment it was omitted.

After tissue and buffer were applied, a 0.010″ thick (0.25 mm) film ofacetate sealing tape for microtiter plates (catalog no. 001-010-3501,Dynatech Laboratories, Chantilly, Va.) with adhesive was applied tocover the samples. The plate was kept on dry ice until use. The platewas loaded onto a x, y, z positioning fixture that was controlled byLabView software. The plate was lowered into the immersion tank filledwith deionized water at room temperature, approximately 22° C. The platewas positioned so that one well was in the focus of the ceramicpiezoelectric dome. The dose was applied through the film and notthrough the bottom of the well. The duration and amplitude, were variedfor the exposure period.

Analysis of material following exposure revealed the exposed leaf tissuesample supernatants were green, whereas control sample supernatants wereonly slightly green, likely due to leaching of chlorophyll from the cutends of the tissue. The treated tissue, when viewed under a dissectingmicroscope, appeared thinner, more translucent, and with small “bubbles”appearing below the initial surface layers. Samples that had beenexposed in the presence of glass beads (Sigma, 212-300 microns, unwashedG-9143, lot No. 75H0617) were not affected as much, based on theobservation that both the buffer solution was clearer and themicroscopic structure of the leaf tissue was different. The leaf tissueappearance was thicker and filled with larger and more “bubbles” belowthe external surface layers. The glass beads may have absorbed orreflected some of the energy applied to the sample. In addition, if thesamples were oriented with the bottom of the polystyrene plate betweenthe dome and the sample, the samples were not noticeably affected,however, with 0.5 V for 20 seconds, melting of the polystyrene bottomoccurred. The polystyrene material may have absorbed the energy in acontinuous wave duration of 20 seconds.

Example 12 Focused Sonolysis with Automated Extraction

Biological material is inserted into a microtube system, where thebottom of the tube is a semi-permeable material such as hydrophobicmembrane. Under atmospheric pressure, the membrane contains bulk waterand under negative pressure allows liquid water and cellularconstituents to traverse. Ideally, the material is transparent toacoustic energy, or at least having similar acoustic properties as thefluid through which the sound energy is transmitted. A leaf tissuesample is placed into a tube with 0.35 ml of CTAB buffer, as in Example1, and frozen. The frozen sample is placed within the focus of a domedultrasonic transducer. The sample is exposed to sonic energy generatedby a 0.5 V signal input to a 55 dB RF amplifier at 1 MHz for 2 seconds.The sample is removed from the exposure chamber and allowed to thaw to4° C. The sample is then vortexed for 10 seconds, and the filtrate isremoved by inserting the tube into a holder and centrifuging the samplefor 10 minutes at 10,000×g. A wash of 0.5 ml of CTAB buffer is appliedto the previously spun sample, the leaf tissue is resuspended in thebuffer, vortexed, and spun as described above. The extract is analyzedfor DNA content.

Example 13 Focused Sound Waves on Frozen Tissue with Real-TimeTemperature Control

100 μg of leaf tissue is frozen in liquid nitrogen. The frozen materialis added directly to a microtube, as described in Example 2, and issuspended in a bath of ethylene glycol chilled to about −15° C.Immediately, 0.25 ml of buffer, chilled to approximately 4° C., isaliquoted into the tube with the tissue. The sample buffer is chilled tobelow 0°. The microtube has been aligned in the focal point of thepiezoelectric transducer. As the sample is chilling, focused sound wavesare applied to the sample. The wavelength, duration, and amplitude aremodulated a priori on test samples to approximate total energy appliedto the system. The system can also utilize a closed-loop feedbackmechanism with an external temperature probe to monitor temperature risein the sample. The sample should be treated with sound waves to inducedisruption, but the temperature should preferably not be elevated aboveabout 0° C.

Example 14 Acoustic Transmission Properties of Polymeric Materials

The acoustic transparency of materials may be measured in order todesign optimal and reproducible shock treatment protocols. To test theacoustic transparency of various polymeric materials, a submergedultrasonic transducer in a water bath was focused on a submergedmicrotiter plate of the type which had wells open at the bottom. Anultrasonic needle-tip hydrophone was placed behind the plate to measurethe transmission across the microtiter plate. The bottom of the platewas blocked with various materials of possible use in the proposedextraction techniques. The transducer was set into a continuous wavegeneration module at low voltage. As shown by the data in Table 7,below, polyethylene terephthalate (PET) material caused the leastattenuation of the acoustic intensity, of these materials. Equivalent orbetter performing materials can be readily identified using routineexperimentation in accordance herewith.

TABLE 7 Relative sonic energy transmission through various materialsRelative transmission of sonic energy (0.05 V at 1 MHz for MaterialThickness of material 10 seconds) No plate 100% Acetate 0.005 inch 80%Latex 0.004 inch 50% PET (Mylar) 0.005 inch 90% Silicone 0.005 inch 95%PET (Mylar) 0.002 inch 95%

Example 15 High Intensity Focused Ultrasound Disruption of Leaf Tissue

Arabidopsis thaliana leaf was collected and immediately immersed inliquid nitrogen. Samples were then stored in a −80° C. freezer untiluse. Leaf samples were inserted into prechilled microtiter plates filledwith approximately 300 ml of refrigerated, precooled CTAB lysis bufferas described in Example 1.

The leaf sample stalk was removed with a single-edge razor blade on aprecooled surface such as a dry ice chilled cutting block. The remainingfrozen tissue was inserted into the microwell and the leaf in the lysisbuffer was either frozen or kept at 4-8° C. until use. The microwellplate was affixed to an x, y, z positioning system to automaticallyalign the samples prior to dosage. The sample plate was previouslyaligned in an insulated bath vessel filled with ethylene glycol that hadan acoustically transparent window on the bottom. The energy systemenables a transducer submerged in a water bath below the sample bathvessel to transmit a focused sound wave through the aqueous transducerbath, then through the acoustic window into the sample bath, and throughthe ethylene glycol liquid in the sample bath to the bottom of themicrowell.

The pulse then entered the CTAB lysis buffer inside the sample well, andwas focused on the leaf tissue. In this example, the focus of the systemwas aligned by applying a low voltage ultrasonic CW and by monitoringbubbling on the surface. The peak focal point was tested to be 2 mm×4 mmand measured with a commercially available needle-point hydrophone thatfit into the microwells.

Using a suitable submersible transducer, energy from the source wasapplied as a continuous wave, generated over approximately 1 MPapositive peak pressure and approximately −1 MPa of negative peakpressure at 50 mV modulation amplitude and at 1 MHz frequency. At thisvoltage, the waveform approximated a symmetrical harmonic. However, asthe voltage increased, the peak positive pressure increased, while thepeak negative pressure became less pronounced. For example, at 700 mV at1 MHz, the peak positive pressure was over 22 MPa (˜3,100 psi) and thepeak negative pressure was only −9 MPa (˜1,300 psi). The repetitivecomplex waveform had sharp positive pressure peaks and blunted negativepressure peaks, due to the nonlinear behavior of the fluid medium(water). The negative pressures are thought to contribute to cavitation.

Using the above apparatus and tuning, three sets of variable doses weregiven to the leaf tissue in microtiter plates. In Series A, pulses wereapplied continuously for 2 million cycles over about a 2 seconds period.At 50 mV, there was no discernible effect on the sample. At 100 and 200mV there was also no effect, but at 500 mV amplitude, the sample wasfull of bubbles and froth, and the extraction buffer turned green withextracted material. When the amplitude was raised to 700 mV, the bottomof the polystyrene plate began to melt. Thus, a certain range of energyintensity is effective in Series A.

In Series B one burst, which was three cycles long, was applied to thesamples. No extraction of material from the leaf disc was observed atany of the voltage levels used, including 700 mV. Thus, a minimum amountof energy is required. Three cycles at these energy levels does notappear to be enough.

In Series C, a 3 cycle burst was applied 100 times. There was no effectat 50, 100 and 200 mV. At 500 mV the solution became slightly green,indicating the beginning of extraction. At 700 mV, the solution becamedefinitely green, indicating substantially complete extraction. Therewas no severe bubbling, or any melting. Thus, it is straightforward todetermine appropriate operating conditions for the use of the extractionsystem of the invention on a particular material in a particulararrangement.

Example 16 Temperature Effects

Using the apparatus of Examples 14 and 15, another series of experimentscompared a slightly above-freezing extraction temperature (6° C. ±2° C.)versus a slightly below freezing temperature (−4° C.+/−1° C.). For allexperiments, the doses were 100 mV, 200 mV, 500 mV, and 0 mV (control)with the 0, 200, and 500 doses in duplicate. The number of bursts wasvaried. The condition of the tissue was observed in this experiment, incontrast to the appearance of the extraction buffer, as in the previousexample.

Series A—above zero temperature

-   -   500 bursts—The control leaf tissue samples (0 mV; no power        applied) were bright green with full appearance. There was no        marked difference in the experimental samples except that they        were slightly more transparent. Near the tip, closest to the        sample source, there appeared to be bubbles connected in        strands.    -   1,000 bursts—the control was bright green with full tissue        appearance. No marked difference was seen among experimental        samples. Again, there was a slight appearance of bubble channels        with slightly more at tips of leaves.    -   5,000 bursts—only the 100 mV experimental appeared close to the        control. All of the other samples had micro channels of linked        bubbles. The samples that had the highest dose had fewer        microchannels than samples receiving lower doses at 5,000        bursts, indicating that acoustic interference with bubble        formation can be a consideration when choosing conditions for        disrupting a particular sample.

Series B—below zero temperature

-   -   500 bursts—Control was intact tissue with no sign of        microchannels. The low dose of 100 mV had a few independent        bubbles, and the 200 mV sample had many independent bubbles (no        microchannels of linked bubbles). Samples in the 500 mV wells        also had independent, discrete bubbles.    -   1,000 bursts—control and 500 mV had slight bubbles. All other        samples had more discrete, independent bubbles.    -   5,000 bursts—the control had slight bubbles formed, but not as        many bubbles as with the 1,000 burst series. The remaining        tissue appeared thin, transparent, and had an apparent loss of        cell structure. The bubbles in the control sample likely        indicated spillover of energy from the adjacent well, which also        was exposed to the most energy. This observation suggests that a        microtiter plate can be made of a material such that the walls        of the wells are capable of absorbing acoustic energy.

Following three days storage between two glass microscope slides at roomtemperature with isolated leaf tissue prepared at subzero temperatures,all of the control tissue appeared green, while the experimental tissuesthat had 100 and 200 mV doses appeared virtually transparent, especiallythe 100 mV samples. The results indicate that substantial disruption ofthe plant leaf tissue occurred as a result of the dosage when theprocess was performed at sub-zero temperatures and the pulse duty cycleminimized sample heating.

Features, specifications, and functionality of the hardware, operatingsoftware, sonic energy profile, and positioning profile of certainembodiments of a system according to the invention are described inFIGS. 10-13. As noted, some of these embodiments can be used effectivelyfor treating a sample for the purpose of extraction or transformation orgeneral research; however, these embodiments are to be consideredexemplary in nature, and not limiting of the invention.

While there has been described herein what are considered to beexemplary and preferred embodiments of the invention, othermodifications and alternatives of the inventions will be apparent tothose skilled in the art from the teachings herein. All suchmodifications and alternatives are considered to be within the scope ofthe invention.

Accordingly, what is desired to be secured by Letters Patent is theinvention as defined and differentiated in the following claims andequivalents thereof.

1. A method for achieving a desired state of fluidization of a sample,comprising: providing a focused acoustic field to a sample, the focusedacoustic field having a frequency of between about 100 kilohertz andabout 100 megahertz and a focal zone having a diameter of less thanabout 2 centimeters; monitoring emissions of acoustic energy from thesample; determining the state of fluidization of the sample based, atleast in part, on the emissions of acoustic energy from the sample; andproviding a modified focused acoustic field to the sample, the modifiedfocused acoustic field having acoustic wave variables selected, at leastin part, based on the determined state of fluidization to achieve thedesired state of fluidization of the sample, wherein the selectedacoustic wave variables include a frequency of between about 100kilohertz and about 100 megahertz and a focal zone having a diameter ofless than about 2 centimeters.
 2. The method of claim 1, wherein thefocused acoustic field has a focal zone smaller than a size of thesample, and the method includes: moving relative positions of the sampleand the focal zone; monitoring the emissions of the acoustic energy fromthe sample at a plurality of relative positions; and determining thestate of fluidization of the sample based, at least in part, on theemissions of acoustic energy from the sample at the plurality ofrelative positions.
 3. The method of claim 1, wherein the sampleincludes a plurality of samples and the providing step includesproviding a plurality of the focused acoustic fields, each to aparticular sample, and the method includes concurrently monitoringemissions of acoustic energy from each particular sample, anddetermining the state of fluidization of each particular sample based,at least in part, on the emissions of acoustic energy from eachparticular sample.
 4. The method of claim 1, wherein the sample includesa plurality of samples and the providing step includes providing aplurality of the focused acoustic fields, each to a particular sample,and the method includes concurrently monitoring reflections of acousticenergy from each particular sample, and determining the state offluidization of each particular sample based, at least in part, on thereflections of acoustic energy from each particular sample.
 5. Themethod of claim 1, wherein the providing step includes providing thefocused acoustic field to a plurality of samples, and the methodincludes concurrently monitoring emissions from each of the plurality ofsamples, and determining the state of fluidization of each of theplurality of samples based, at least in part, on the emissions from eachof the plurality of samples.
 6. The method of claim 1, including flowingthe sample through the focused acoustic field.
 7. The method of claim 1,including providing the focused acoustic field to a reaction vesselcontaining the sample.
 8. The method of claim 1, including concurrentlymonitoring an optical response signal from the sample, and determiningthe state of fluidization based, at least in part, on the opticalresponse signal.
 9. The method of claim 8, wherein the optical responsesignal includes at least one of reflective, absorptive and fluorescentspectral content.
 10. The method of claim 1, including concurrentlymonitoring temperature information from the sample, and determining thestate of fluidization based, at least in part, on the temperatureinformation.
 11. The method of claim 1, wherein the sample includesorganic material.
 12. The method of claim 1, wherein the sample includesinorganic material.
 13. The method of claim 1, wherein the sampleincludes a mineral.
 14. The method of claim 1, wherein the sampleincludes a biological material.
 15. The method of claim 1, includingsuspending the sample in a fluid.
 16. The method of claim 15, whereinthe fluid includes a solvent.
 17. A method for achieving a desired stateof mixing of a sample with a constituent comprising: providing a focusedacoustic field to a sample, the focused acoustic field having afrequency of between about 100 kilohertz and about 100 megahertz and afocal zone having a diameter of less than about 2 centimeters;monitoring acoustic energy from the sample; determining the state ofmixing of the sample with the constituent based, at least in part, onthe acoustic energy from the sample; and providing a modified focusedacoustic field to the sample, the modified focused acoustic field havingacoustic wave variables selected, at least in part, based on thedetermined state of mixing to achieve the desired state of mixing of thesample, wherein the selected acoustic wave variables include a frequencyof between about 100 kilohertz and about 100 megahertz and a focal zonehaving a diameter of less than about 2 centimeters.
 18. The method ofclaim 17, wherein the acoustic energy from the sample includes reflectedacoustic energy.
 19. The method of claim 17, wherein the acoustic energyfrom the sample includes emitted acoustic energy.
 20. The method ofclaim 17, wherein the focused acoustic field has a focal zone smallerthan a size of the sample, and the method includes: moving relativeposition of the sample and the focal zone; monitoring the acousticenergy from the sample at a plurality of relative positions; anddetermining the state of mixing of the sample with the constituentbased, at least in part, on the acoustic energy from the sample at theplurality of relative positions.
 21. The method of claim 17, wherein theproviding step includes providing a plurality of the focused acousticfields, each to a particular sample and associated constituent, and themethod includes concurrently monitoring acoustic energy from eachparticular sample, and determining the state of mixing of eachparticular sample with its associated constituent based, at least inpart, on the acoustic energy from each particular sample.
 22. The methodof claim 17, wherein the providing step includes providing the focusedacoustic field to a plurality of samples, and the method includesconcurrently monitoring acoustic energy from each of the plurality ofsamples, and determining the state mixing of each of the plurality ofsamples with an associated constituent based, at least in part, on theacoustic energy from each of the plurality of samples.
 23. The method ofclaim 17, including the step of flowing the sample and the constituentthrough the focused acoustic field.
 24. The method of claim 17,including providing the focused acoustic field to a reaction vesselcontaining the sample and the constituent.
 25. The method of claim 17,including concurrently monitoring an optical response signal from thesample, and determining the state of mixing of the sample with theconstituent based, at least in part, on the optical response signal. 26.The method of claim 25, wherein the optical response signal includes atleast one of reflective, absorptive and fluorescent spectral content.27. The method of claim 17, including concurrently monitoringtemperature information from at least one of the sample and theconstituent, and determining the state of mixing of the sample with theconstituent based, at least in part, on the temperature information. 28.The method of claim 17, wherein the constituent includes a solvent. 29.The method of claim 17, wherein sample includes a first compound and theconstituent includes a second compound, different from the firstcompound.
 30. The method of claim 17, wherein the sample includes anantibody and the constituent includes a molecule to which the antibodybinds.
 31. The method of claim 17, wherein the sample includes asubstrate and the constituent includes a ligand.
 32. The method of claim17, wherein the sample includes at least one of an antibody and areceptor and the constituent include a support surface for immobilizingthe at least one of the antibody and the receptor.
 33. The method ofclaim 17, wherein the sample includes a first nucleic acid and theconstituent includes a second nucleic acid, different from the firstnucleic acid.
 34. The method of claim 33, wherein the first nucleic acidmolecule is a primer and the second nucleic acid is a substrate.